Sol-gel polymer composites and uses thereof

ABSTRACT

The present disclosure relates generally to sol-gel polymer composites that comprise chitosan, a hydrophilic polymer, a gelation agent, and optional additional ingredients in a suitable medium. Advantageously, the sol-gel polymer composite can form a durable seal or strong solid in response to one or more physiological stimulus. The disclosure further relates to medical and veterinary uses of the composite, particularly, methods and delivery systems for reducing or preventing the incidence of a mammary disorder in a dairy animal. More particularly, the disclosure includes methods and sol-gel polymer composite compositions for creating a physical barrier on the teat surface or in the teat canal or cistern of a non-human animal for prophylactic treatment of mammary disorders such as mastitis wherein the sol-gel polymer creates a seal in response to one or more physiological stimulus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Nos. 62/250,126 and 62/315,756, filed Nov. 3,2015 and Mar. 31, 2016, respectively. The prior applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates generally to unique sol-gel polymercomposites and novel uses for them. More particularly, the disclosurerelates to the sol-gel polymer composites that form a strong solid inresponse to a physiological stimulus, the strong solid havingpre-determined permeability and mechanical properties in response to thephysiological stimulus. The composites are easily injectable and haveshear thinning properties making them useful in a wide range of humanand animal health applications where it is desirable to inject a liquidthat solidifies rapidly after injection in a subject. The disclosurealso relates to new methods for protecting the mammary glands of dairyanimals from pathogenic load by utilizing the composites as teatsealants to decrease or to prevent the incidence of mastitis in theanimals.

BACKGROUND

The contents of all patents and publications cited in this specificationare hereby incorporated by reference in their entirety.

Hydrogels are highly hydrated, macromolecular networks, dispersed inwater or other biological fluids. Hydrogels that exhibit the specificproperty of increased viscosity with increased temperatures are known asthermoreversible, thermosensitive (or thermosetting) hydrogels. It isknown that thermosensitive hydrogels may be prepared from polymers ofnatural origin such as chitosan, which is a commercially available,inexpensive polymer obtained by partial to substantial alkalineN-deacetylation of chitin, a linear polysaccharide, made ofN-acetylglucosamine units, linked via β-1,4-glycosidic bonds. Thedeacetylation process is generally performed using hot, concentrated,hydroxide solutions, usually sodium hydroxide.

Chitosan is biocompatible, non-toxic, and non-immunogenic, allowing itsuse in the medical, pharmaceutical, cosmetic and tissue constructionfields. For example, topical ocular applications and intraocularinjections or transplantation in the vicinity of the retina have beenused. Moreover, chitosan is metabolized-cleaved by certain specificenzymes, e.g., lysozyme, and can therefore be considered asbiodegradable. In addition, it has been reported that chitosan acts as apenetration enhancer by opening epithelial tight junctions. Chitosanalso promotes wound healing and exhibits antibacterial, antifungal andantitumor properties.

The complexity of biological structures such as natural tissue hasresulted in researchers exploring the use of biomaterials and medicaldevices that are introduced on the skin or into the body of a subject asa liquid and that turn solid or solid-like through simple application orinjection. For example, chitosan hydrogels have been shown to be usefulfor cartilage regeneration and prevention of knee pain associated withacute and chronic cartilage defects. Chitosan-based gels have also beenshown to turn into and serve as scaffolds for the encapsulation ofinvertebral disc (IVD) cells by entrapping large quantities of newlysynthesized anionic proteoglycan. Chitosan is known to formthermoreversible gels in the presence of several multivalent anions,such as phosphate derivatives. Temperature-controlled pH-dependentformation of ionic polysaccharide gels, such aschitosan/organo-phosphate aqueous systems, has been described, forexample, in PCT International Publication No. WO 99/07416 and U.S. Pat.No. 6,344,488. However, hydrogels made from ionic polysaccharides suchas chitosan are weak and usually form only after a relatively longwaiting time, after mixing polymer and salt solution. This is mainly dueto the fact that it is difficult to obtain homogenous, fully-hydratedchitosan solutions with a high concentration of chitosan, especiallyhigh molecular weight chitosan, due to its poor solubility. Further,several medical applications require provision of not only a simplesol-gel transition, but a solid structure with desired macroporosity andmechanical properties. Moreover, temperature is a non-specific stimulusand can be triggered by elements outside the human body such as hotweather or, for oral applications, simply drinking a hot beverage. Thus,there is a need for stimuli-responsive implants and patches that canreach desired mechanical and/or permeability properties only whentriggered by specific physiological stimuli.

U.S. Pat. No. 9,034,348 discloses injectable chitosan mixtures forminghydrogels. There are described chitosan compositions which form ahydrogel at near physiological pH and 37° C., comprising at least onetype of chitosan having a degree of acetylation in the range of fromabout 30% to about 60%, and at least one type of chitosan having adegree of deacetylation of at least about 70%. Further disclosed is achitosan composition which forms a hydrogel at near physiological pH and37° C. that includes at least one type of chitosan having a degree ofdeacetylation of at least about 70% and a molecular weight of from10-4000 kDa, and at least one type of a chitosan having a molecularweight of from 200-20000 Da. Also disclosed are methods of preparationand uses of the chitosan compositions.

U.S. Patent Application Publication No. 2010/0028434 disclosestemperature controlled and pH-dependent self-gelling biopolymericaqueous solutions. There are described biopolymeric liquid aqueouscompositions for producing self-gelling systems and gels, whichcomprises an acidic water-based medium, 0.1 to 10% by weight of apH-gelling acid-soluble biopolymer, and 0.1 to 10% by weight of awater-soluble molecule having a basic character and a pKa between 6.0and 8.4, or a water-soluble residue or sequence of the molecule having abasic character and a pKa between 6.0 and 8.4. The liquid compositionshave a final pH ranging from 5.8 and 7.4, and form a stable solid andhomogeneous gel within a temperature range from 10 to 70° C. Methods forpreparing the compositions and uses thereof are also described.

U.S. Patent Application Publication No. 2010/0285113 discloses inversethermal gelling composite hydrogels having enhanced stability. There aredescribed composite hydrogels comprising a blend of an aqueous solutionof an anionic polysaccharide or a derivative thereof, such as hyaluronan(also commonly referred to as hyaluronic acid) or a derivative thereofand an aqueous solution of methylcellulose or another water solublecellulose derivative thereof, having dispersed polymeric particles, suchas polymeric hydrophobic particles therein selected from microparticlesand nanoparticles, and wherein the stability of the hydrogel is enhancedrelative to the stability of the hydrogel alone. The polymeric particlesmay contain at least one therapeutic agent, in which case eachtherapeutic agent exhibits a linear sustained release rate that can betuned or altered by selecting the appropriate polymer formulation of themicroparticles and/or nanoparticles. The composite may be injectable,and in the absence of a therapeutic agent may be used as a bulking agentfor reconstructive and cosmetic surgery or may act as a platform forsubsequent delivery of therapeutic agents.

Insofar as veterinary health issues are concerned, mastitis is aninflammation of the mammary gland that is typically caused by bacteriawhich in most cases enter the gland via the teat orifice. During thenon-lactating period or “dry period” in the gland, deposits of keratinin the teat orifice and the streak canal form a primary defensemechanism. A keratin plug that forms in the teat of the animal forms aprotective barrier, and the immune-rich tissues of the Furstenburg'sRosette in the teat, as well as the natural protective factors of thedry-cow secretions, contain high levels of naturally occurringanti-bacterial substances (cationic proteins) which inhibit the passageof bacteria from the teat orifice to the teat cistern (papillary sinus)and gland cistern. However, this keratin plug and these natural immunedefense mechanisms can be overcome by bacterial invasion as the animalenters into the dry period at the end of lactation, during the dryperiod of the animal, and/or during calving. As a result, bacteriainvade the gland and cause mastitis during the dry period or, moreparticularly, immediately following calving.

The major pathogens causing mastitis are Staphylococcal species such as,for example, Streptococcus agalactiae, Staphylococcus aureus and thelike, Corynebacterium bovis, Mycoplasma, coliforms such as, for example,Esherichia coli, Klebsiella spp., Enterobacter spp., and Citrobacterspp., environmental Streptococcal species such as, for example, Strep.dysgalactiae, Strep. uberis, and Enterococcus spp., Pseudomonas spp.,etc. Although mastitis is mainly caused by bacteria, the inflammationcan also be produced as a result of viral infection (e.g., bovineherpesvirus II and IV, a paravaccinia virus such as Pseudo Cowpox, andthe like) or infection with atypical pathogens like mycotic (e.g.,Candida spp. and Aspergillus spp.) or algal microbes (e.g., Protothecaspp.) with or without development of a secondary bacterial infection.

Mastitis due to the presence of pathogens can become a highly contagiouscondition within the confines of a dairy farm that results in hugeproduction losses for the dairy industry. Reduction of drinkable milkthen occurs from the harmful pathogens' effects or various treatmentsthat render the milk not fit for human consumption. While severe casescan end in death, unhindered outbreaks can also cause permanent damageto the animals' udders. As a major endemic disease of dairy animals,mastitis puts the animal welfare at risk and often entails rather costlyveterinary care. The value of protecting the early lactation period fromexisting and new infections perpetuated from the dry period remainshighly valuable to the industry. It is clear that the treatment andcontrol of mastitis is an important goal to maintain the animal's healthand to lower the high costs of milk production in the dairy industry.

To that end, products have been developed in an attempt to seal ananimal's teat to prevent mastitis and other conditions, for example,barrier teat dips to seal the external surface and streak canal of theteat during periods of milking and internal teat sealants to block or toseal the teat canal or to plug the teat cistern during the dry period,especially for heifers and cows that have experienced one or morepregnancies previously.

Along with these products, several methods to reduce the incidence ofmastitis are described in the art, for example, a method comprisingsequentially delivering from a single delivery device an antimicrobialformulation and a seal formulation into the teat canal of a non-humananimal wherein the seal formulation is nontoxic heavy metal salt such asbismuth (U.S. Pat. No. 8,353,877); a method of applying to the teatcanal and/or teat sinus a composition comprising exogenous keratin (U.S.Pat. No. 8,226,969); a method of forming a physical barrier in the teatcanal for prophylaxis during an animal's dry period by infusing anamount of a teat seal formulation into the teat canal of the animal,wherein the teat seal formulation comprises a bismuth-free, nontoxic,heavy metal salt of titanium, zinc, barium or combinations thereof andthe physical barrier does not cause a black spot defect in dairyproducts made with milk from the animal (U.S. Pat. No. 7,906,138); amethod of forming an anti-infective free physical barrier in theanimal's teat canal for prophylactic treatment of mastitis during thedry period comprising the step of infusing a seal formulation into theteat canal of the animal without an anti-infective agent, wherein theseal formulation comprises a nontoxic heavy metal salt such as bismuthin a gel base of aluminum stearate with a vehicle such as liquidparaffin or a gel base comprising a polyethylene gel (U.S. Pat. No.6,254,881) and the like.

However, none of the existing seal formulations or external dip productsseals the teat of the dairy animal externally for a sufficient amount oftime to prevent mastitis, particularly the form that can be fatal and/orvery contagious in the animals, like among heifers. Moreover, while teatsealants have been established as a viable method to provide a higherlevel of protection regardless of antibiotic choice or administration,the current products on the market fail to meet the demand for ease ofuse and long-lasting tissue adherence, ease of removal, avoidance ofmilk contamination and prevention of black spot defect in aged cheese.What is needed, therefore, is a nontoxic formulation that is easy andsafe for the animal handler to administer and that preferably forms aneffective, long-lasting seal in place directly on the tissue (that is,“in situ”). Additionally, it is necessary for the seal formulation notto interfere with the quality of the dairy animal's milk, yogurt orcheese products created from the milk, especially for the sealant toavoid the black spot defect in aged cheese. Indeed, there is a definiteart-recognized need in the veterinary field to find a long-lasting,nontoxic, non-irritating seal formulation that forms an adequate barrieron the animal's teat to prevent or to reduce significantly the incidenceof mastitis caused by pathogens, preferably without the use ofantibiotics or other medicinal agents that require a withholding periodfor public consumption of the animal's milk. There is also a definiteneed to find a long-lasting seal formulation that can containantibiotics and the like for the effective treatment or prevention ofmastitis.

It is an object of the present technology, therefore, to provide sol-gelpolymer composites that ameliorate the inconveniences of the knownhydrogels.

BRIEF SUMMARY

The present disclosure concerns new sol-gel polymer composites thatcomprise chitosan, a hydrophilic polymer, and a gelation agent in asuitable medium. Advantageously, the sol-gel polymer composite can forma durable seal or strong solid in response to one or more physiologicalstimulus. This disclosure further concerns a variety of medical andveterinary uses for the sol-gel polymer composites. Specifically, thedisclosure involves new methods of forming a physical barrier in theteat canal of a dairy animal for the prophylactic treatment orprevention of mammary disorders that occur mainly as the animal entersthe dry period or during the dry period, comprising the basic step ofexternally applying a sol-gel polymer composite to the teat of theanimal or infusing the composite within the teat canal or cistern.Preferably, the composition gels or solidifies rapidly in response toone or more physiological stimulus to form a strong solid. Thisdisclosure also provides systems for forming a physical barrier in theteat canal of a dairy animal for the treatment of mammary disorders,said system comprising a sol-gel polymer composite and an infusiondevice for infusing the composition into the teat cistern of the animal.Such systems permit the teat sealant to block the invasion of themammary gland by a mastitis-causing microorganism or to decrease theoccurrence or re-occurrence of infection.

BRIEF DESCRIPTION OF THE DRAWINGS

The background of the disclosure and its departure from the art will befurther described herein below with reference to the accompanyingdrawings, wherein:

FIG. 1A shows graphs of time dependence of elastic modulus (G′) and losstangent (tan δ=G″/G′) upon a temperature jump from 25 to 37° C. recordedat f=0.1 Hz and oscillatory stress of τ=1 Pa, for the F1-20141210formulation.

FIG. 1B shows graphs of time dependence of elastic modulus (G′) and losstangent (tan δ=G″/G′) upon a temperature jump from 25 to 37° C. recordedat f=0.1 Hz and oscillatory stress of τ=1 Pa, for the F4-20141210formulation.

FIG. 2 shows a graph of time dependence of elastic modulus (G′) and losstangent (tan δ=G″/G′) upon a temperature jump from 25 to 37° C. forF1-20141210 formulation recorded at time 0 and 12 weeks aftersterilization at f=0.1 Hz and oscillatory stress of τ=1 Pa.

FIG. 3 shows a graph of stress sweep results for the formulationsF1-20140825 (nonsterile) and F1-20141210 (sterile and stored for 12weeks at room temperature) registered at oscillation frequency f=0.1 Hzand temperature T=25° C. The arrow indicates the onset of theshear-thinning region, which was similar for both samples.

FIG. 4 shows a graph of dependence of the gelation temperature on theamount of nanocrystalline cellulose (NCC) for a formulation of 17% w/wPluronic® F127 in 1% w/w chitosan (CH) (pH of around 6).

FIG. 5 shows a graph of viscosity of PVA-acylate at 6 s⁻¹ as a functionof time and at different temperatures.

FIG. 6 shows stress sweep results for sol-gel polymer compositeformulations F1-F4, as indicated, at oscillation frequency f=0.1 Hz andtemperature T=25° C. The arrows indicate the beginning of theshear-thinning region.

FIG. 7 shows time dependence of elastic modulus G′ and loss tangent, tanδ=G″/G′, upon a temperature jump from 25 to 37° C. recorded at f=1 Hzand oscillatory stress of τ=1 Pa.

FIG. 8 shows the average release rate of amoxicillin from sol-gelpolymer composite formulation F2 at T=25° C. and T=37° C. Error barsrepresent standard deviation (n=3).

DETAILED DESCRIPTION OF THE DISCLOSURE

In accordance with the present disclosure, there are provided novelsol-gel polymer composites which comprise chitosan, a hydrophilicpolymer, and a gelation agent in a suitable medium, desirably an aqueousmedium and more desirably, in a weakly acidic, aqueous-based medium. Thesol-gel polymer composite has shear thinning properties such that thecomposite can be deformed in a syringe at room temperature. The sol-gelpolymer composite is also capable of being injected using asingle-barrel syringe and the like. Beneficially, the composite iscapable of forming a solid, often an especially strong solid, inresponse to one or more physiological stimulus without the addition ofany other agents. In the response to stimulus, the sol-gel polymercomposite often solidifies rapidly. Another advantage is that thecomposite provides an instant-gelling strong solid capable ofwithstanding mechanical or hydraulic pressures in physiologicalconditions. Additionally, the composite is capable of forming a no-leak,no-drip plug after injection into a mammalian subject.

The disclosure further provides numerous medical and veterinary uses forthe sol-gel polymer composites that benefit from the specially designedformulations. While human applications will become apparent from thedisclosure, a preferred use relates to a unique method of forming aphysical barrier in the teat canal of a dairy animal for theprophylactic treatment of mammary disorders that typically occur as theanimal begins to dry off or during the dry period, comprising the stepof administering a sol-gel polymer composite to the teat or within theteat canal of the animal, preferably embracing the composition that gelsor solidifies rapidly in response to one or more physiological stimulusto form a durable seal or strong solid. Also, the disclosure providesnew methods of treatment that block the invasion of the mammary gland bymastitis-causing microorganisms and reduce or prevent the incidence ofnew infections or re-infection.

This disclosure includes systems for forming a physical barrier, whichis preferably an internal barrier within the teat canal, of a dairyanimal to prevent mammary disorders or to lessen the harmful effects ofinfection, said system comprising a sol-gel polymer composite and adelivery device for infusing the composition into the teat cistern ofthe animal. Such systems permit the treatment to block the invasion ofthe mammary gland by a mastitis-causing microorganism or to decrease therisk of the occurrence or re-occurrence of infection. More particularly,the present disclosure provides methods and systems wherein the sol-gelpolymer composite is infused predominantly as the animal begins to dryoff or during the dry period of a dairy livestock animal, preferably aheifer or a cow, but also can include other animals such as goats,sheep, water buffaloes and the like. The sol-gel polymer composite actsas an aid in the prevention and the control of mastitis during the dryoff period, thus reducing the clinical and sub-clinical cases during thedry off period and in the first stage (post calving) of lactation. Byremaining in the teat canal throughout the dry period, the sol-gelpolymer composite eliminates or reduces microbial invasion through theteat canal during high risk periods in the pre-fresh dairy animal.

In one aspect, the disclosure provides methods for combatting microbialmammary mastitis in a dairy animal which method permits milk obtainedfrom the animal to be used in the production of a milk product, themethod comprising applying topically or infusing a sol-gel polymercomposite directly on the relevant mammary tissue of a dairy animal orwithin the teat canal to form a teat seal. This teat sealant wouldtypically be administered via intramammary administration to each teatat the time of drying-off. Preferably, the sol-gel polymer composite isapplied or infused prior to infection of a healthy animal. In anotheraspect, the disclosure provides methods for reducing the withholdingtime of milk obtained from an animal being treated for mastitis beforepublic consumption is allowed in the production of a milk product,wherein the sol-gel polymer composite is applied topically to or infusedwithin the teat canal of the animal. The present disclosure alsoprovides methods for reducing the withholding time of milk obtained froman animal being prophylactically treated to prevent or to reduce thefrequency of mastitis in order to improve the production of a milkproduct, wherein a sol-gel polymer composite is applied topically to orinfused within the teat canal of the animal.

Even more particularly, the present disclosure provides the abovemethods wherein the dairy product is milk, yogurt or cheese. When thedairy product is milk, the methods encompass dry or fluid milk. Also,the disclosure provides such methods wherein the sol-gel polymercomposite is administered via intramammary infusion or by dipping theteat. In all embodiments, the non-human animal in need of the relevantveterinary uses of the present disclosure is preferably a heifer or cowbut also can be another dairy livestock animal; and the administrationis preferentially achieved by intramammary infusion as the animal beginsto dry off or during the dry period. Moreover, the disclosure providessuch methods wherein the sol-gel polymer composite is administeredduring the postpartum period of a non-lactating animal or wherein thesol-gel polymer composite is administered during the prepartum period ofan animal.

The teat sealant of the present disclosure provides many advantages overthe current sealants on the market through enhanced ease of use (bothadministration and removal) as well as in the total sum of its novelquality profile, for instance, the sol-gel polymer composite'snontoxicity, biocompatibility, biodegradability, elasticity(pliability), long-lasting tissue adherence, syringability, fluidity atroom temperature, ability to solidify in response to body temperature,fast gelation time at 37° C., nonirritating and inert nature, etc.Notably, the infusible aqueous-based, thermal-transition sol-gelhydrogels are uniquely fluid at room temperature yet form a gel at bodytemperature in the teat canal. The sol-gel polymer formulationsdemonstrate a tailorable shear thinning characteristic which allows forease of infusion over a wide temperature range as well as removal bymanual stripping from the teat canal. Upon removal from the body cavity,the sol-gel polymer composite returns to a liquid phase at roomtemperature which supplies a real benefit to the dairies. In addition,unlike current commercial sealants, the teat sealant of this disclosuredoes not stick to the stainless steel pipes (milk lines) or bulk tanksduring the initial processing stage of milk and is cleanable (that is,will readily clean off the industrial surfaces) during standard cold orhot water washes, which ultimately avoids milk contamination therebypreventing the black spot defect often seen in aged cheese and caused byknown, conventional teat sealants.

Also beneficially, the teat sealant of the disclosure eliminates orsignificantly reduces the withholding time of milk obtained from anon-human animal being treated prophylactically for mastitis therebyavoiding or decreasing the standard milk discard period that is requiredwhen the animals are given antibiotics. Equally practical, the teatsealant can be preferentially designed to be fully compatible withcheese starter cultures when prepared with a relatively neutral pH byvarying the salt component of the sol-gel polymer composite. Colostrumfrom treated animals is also safe to feed to calves.

DEFINITIONS

It should be appreciated that all scientific and technological termsused herein have the same meaning as commonly understood by those ofordinary skill in the art. The following definitions are given merely toillustrate the general meanings of the main terms used in connectionwith the present disclosure.

The term “udder” refers herein to the glandular, mammary structure of afemale ruminant animal such as a cow, a goat, a sheep, a water buffaloand the like. In the cow, it comprises four independent glands, with oneteat and one exit duct each, whereas sheep and goat have two glands. Theterm “teat” refers herein to the projecting part of the mammary glandcontaining part of the milk or teat sinus.

The term “teat sealant” refers herein to compositions and devices usedto form a physical barrier on the surface of or inside an animal teat. Ateat sealant can be on the teat surface, inside the teat streak canal,and/or inside the teat cistern.

The term “antimicrobial” refers herein to a substance that kills orinhibits the growth or reproduction of microorganisms such as bacteria,viruses, fungi, yeast, or protozoans.

The term “solution” refers herein to solutions, suspensions, ordispersions, unless otherwise stated. The term “spray” as used hereinrefers to an atomized composition, such as comprised of small or largeliquid droplets, such as applied through an aerosol applicator or pumpspray applicator for the intended purpose of delivering a broadapplication of the composition.

The term “stream” refers herein to a continuous, direct, and focusedapplication of the composition. The term “infusion” refers herein to thecontinuous introduction of a fluid or solution into a cavity, vein orcistern.

The term “mammal” refers herein to a warm-blooded vertebrate animal ofthe class Mammalia, which includes both human and animal, that possesshair or fur on the skin, the secretion of milk from milk-producingmammary glands by females for nourishing the young, and a four-chamberedheart.

For the embodiments of the disclosure that relate to mastitis, the term“animal” refers herein to a female, non-human mammal which has alactation period, which includes, but is not limited to, livestockanimals, such as cows, sheep, goats, horses, pigs, water buffaloes andthe like. Preferably, the animal is a dairy cow. While both the “cow”and the “heifer” are female bovines, the term “heifer” refers herein toany young female cow that has not given birth to a calf, typically onethat has been weaned and under the age of 3 years. The term “cow” oftenrefers to an older female animal that has given birth to a calf.

The term “dry period” refers herein to the non-lactating phase of thelactation cycle of a cow or other dairy animal. It occurs between theend of one lactation cycle and the beginning of the next lactation. Atthe end of each lactation cycle, the animal begins the phase of “dryingoff” as the animal enters the dry period which includes the usualphysiological, metabolic and endocrine changes associated with cessationof milk production for the non-lactating period (dry period) of theanimal.

The term “milk product” refers herein to a product containing any amountof milk in liquid or powder form. It also includes cheese and yogurt.

The term “postpartum” refers herein to the period of time beginningimmediately after calving and extending for about six weeks. The term“prepartum” refers herein to the period of time during pregnancy, whichis prior to calving. The term “periparturient” refers herein to theperiod immediately before and after calving.

The term “involution” refers herein to the first two to three weeksafter cessation of milk production in a cow.

The term “keratin plug” refers herein to keratin-based occlusion of theteat canal/streak canal of a cow following cessation of milk productionfor the dry period.

The term “microbial invasion” refers herein to movement of pathogenicmicroorganisms such as, for example, bacteria, especially pus-forming ornecrotizing bacteria, viruses, fungi, yeast, protozoans and the likethat proliferate into bodily tissue or bodily cavities, resulting intissue injury that can progress to infection and/or disease. Forpurposes of the disclosure, the “microbial invasion” typically refersherein to a “bacterial invasion.”

The term “sol-gel polymer composite” refers herein to a polymercomposition that can undergo a sol-gel process to form a sol-gel stateunder certain conditions, as described herein. The terms “solid” and“gel,” and “solidification” and “gelation” are used interchangeableherein to refer to the gel/solid formed after the sol-gel phasetransition has occurred in response to one or more physiologicalstimulus.

The term “polymer” refers herein to a material that includes a set ofmacromolecules. Macromolecules included in a polymer can be the same orcan be differ from one another in some fashion. A macromolecule can haveany of a variety of skeletal structures, and can include one or moretypes of monomeric units. In particular, a macromolecule can have askeletal structure that is linear or non-linear. Examples of non-linearskeletal structures include branched skeletal structures, such thosethat are star-branched, comb-branched, or dendritic-branched, andnetwork skeletal structures. A macromolecule included in a homopolymertypically includes one type of monomeric unit, while a macromoleculeincluded in a copolymer typically includes two or more types ofmonomeric units. Examples of copolymers include statistical copolymers,random copolymers, alternating copolymers, periodic copolymers, blockcopolymers, radial copolymers, and graft copolymers.

As used herein with reference to a polymer, the term “molecular weight(MW)” refers to a number average molecular weight, a weight averagemolecular weight, or a melt index of the polymer.

The term “elastic modulus” (also referred to as “Young's modulus” or thestorage modulus (G′)) is defined herein as the change in stress with anapplied strain (that is, the ratio of shear stress (force per unit area)to the shear strain (proportional deformation)) in a material.Essentially, the elastic modulus is a quantitative measurement ofstiffness of an elastic material that measures the ability of the testedmaterial to return to its original shape and size. G′ can be calculatedusing a formula derived from Hooke's law, which states that the elasticmodulus is equal to the ratio of stress to strain (i.e., the ratio ofapplied pressure to fractional change in size). The measure of theelastic modulus is reported as the force per unit area (the standardmetric ratio of the Newton to unit area (N/m²) or the pascal (Pa) inwhich one pascal is equivalent to one Newton (1N) of force applied overan area of one meter squared (1 m²)). This pascal unit is anart-recognized term often used to define a unit of pressure, tensilestrength, stress and elasticity.

The term “shear thinning” as used herein refers to the commoncharacteristic of non-Newtonian fluids in which the fluid viscositydecreases with increasing shear rate or stress. Shear thinning isobserved in suspensions, emulsions, polymer solutions and gels. Due toshear thinning attributes, decreasing the viscosity of a polymer, amacromolecule or gel is made possible by increasing the rate of shear.Basically, as a result of the decrease in viscosity upon increase inshear rate, the “shear thinning” property is a measure of the ability ofthe hydrogel network to be temporarily deformed through the applicationof a gentle manual pressure from the piston of a syringe. This shearthinning phenomenon may be used, for instance, to make an otherwisestiff biocompatible hydrogel infusible.

The term “loss tangent tan δ” or “tan δ” refers herein to the tangent ofthe phase angle, that is, the ratio of viscous modulus (G″) to elasticmodulus (G′) and a helpful quantifier of the presence and the degree ofelasticity in a fluid. The tan δ values of less than unity indicateelastic-dominant (i.e. solid-like) behavior and values greater thanunity indicate viscous-dominant (i.e. liquid-like) behavior. In anelastic solid, tan δ″=0.

As used herein, “strong” is intended to mean the elastic modulus G′ thatcan generally range widely from about 420 Pa or higher, about 600 Pa toabout 10,000 Pa, or about 6000 Pa to about 10,000 Pa, etc. atphysiological temperature. Based on the level of stiffness, a solidbody, for example, deforms when a load is applied to it. If the materialis elastic, the body returns to its original shape after the load isremoved. A “strong solid” is generally a gel or solid formed after thesol-gel phase transition for which G′ at physiological conditions (e.g.,37° C., and/or near physiological pH) is typically above about 560 Pa,although strong solids may form below about 560 Pa or above about 10,000Pa depending on other factors in the processing steps to make, tosterilize or to store the formulation.

The term “physiological temperature” used herein is intended to mean thenormal body temperature range for a mammal, e.g., about 35° C. to about40° C., about 36° C. to about 40° C., about 37° C., about 37.5° C. andthe like.

The term “one or more physiological stimulus” refers herein to aselection of one or more stimulus embracing, but not limited to,temperature (e.g., body temperature such as a temperature from about 36°C. to about 40° C., or about 37° C.), pH (e.g., near physiological pH,alkaline or acidic conditions), ionic strength (e.g., hypotonic orhypertonic conditions) and the like. Other types of physiologicalstimuli include exposure to a bodily fluid such as, for example, breastmilk or other secretions, blood, and the like. Another type of stimulimay arise from contact with a bodily chemical or macromolecule such aswithout limitation ions, electrolytes, calcium, sodium, cytotoxins,macrophages, enzymes, antigens, glucose, estrogen, etc.

Components and Characteristics of the Composition

In general, the sol-gel polymer composite of the present disclosurecomprises chitosan, a hydrophilic polymer, and a gelation agent in asuitable acidic water-based medium. Optionally, the sol-gel polymercomposites further include a reinforcing agent such as suitablenanocrystalline fillers and/or one or more antimicrobial agents.Advantageously, the sol-gel polymer composite forms a durable seal or astrong solid in response to one or more physiological stimulus. Theformulations exhibit a unique combination of deliverability, swelling,and adhesion.

For the elements of the new sol-gel polymer composite, the chitosan isacylated in some instances, for example, the chitosan comprises acylchitosan which includes, but is not limited to, carboxymethyl chitosan(CMCh). In some embodiments, the chitosan has a degree of deacetylation(% DDA) of at least about 75%, at least about 77%, at least about 80%,or at least about 90%. In some embodiments, the chitosan has a % DDA ofabout 75%, about 77%, about 80%, about 95%, about 96%, about 97%, about98%, about 99%, or higher. The hydrophilic polymer includes, but is notlimited to, methyl cellulose (MC) such as methyl cellulose ethers orcellulose ethers, polyvinyl acetate (PVA), PVA-acylate, hydroxypropylcellulose (HPC), ethyl hydroxyethyl cellulose (EHEC), hyaluronic acid(HA), a poloxamer (a nonionic triblock copolymer) such as Pluronic®,polyethylene glycol (PEG), sodium alginate, or another water-solublepolysaccharide capable of forming a highly viscous thermosensitive gel.The hydrophilic polymer may be acylated. Desirably, the sol-gel polymercomposite comprises methyl cellulose or PVA-acylate, which forms a thinmixture (slurry) after dissolution in cold water and a thick gel atphysiological temperatures.

Usually, the gelation agent is a thermogelling element that undergoesphysical crosslinking in response to a stimulus, e.g., temperature. Insome embodiments, the gelation agent is a salt, such as,β-Glycerophosphate disodium hydrate or pentahydrate, sodiumpyrophosphate tetrabasic, potassium phosphate dibasic trihydrate,mixtures thereof and the like. Advantageously, the gelation agent is amixture of sodium pyrophosphate tetrabasic and potassium phosphatedibasic trihydrate salts. The terms “gelation agent” and “gelator” areused interchangeably herein.

It is preferable to prepare the sol-gel polymer composites in a weaklyacidic aqueous-based medium such as, for example, 0.1M aqueous aceticacid.

The formulation of the sol-gel polymer composites may optionallyencompass a reinforcing agent such as a nanocrystalline filler. Thestrengthening material referred to as the “nanocrystalline filler” isgenerally a nanocrystalline material, e.g., a nanocrystalline particleor polymer, capable of providing mechanical reinforcement to the sol-gelpolymer composite through noncovalent physical interactions such as,without limitation, hydrogen bonds or electrostatic attractions.Examples include, but are not limited to, nanocrystalline cellulose(NCC), an inorganic clay, an organic clay, carbon black, fumed silica,graphene, graphite and the like. Preferably, the nanocrystalline filleris nanocrystalline cellulose (NCC). Alternatively, the nanocrystallinefiller comprises, for instance, a nanocrystalline starch, nanoclay,graphene, a carbon nanotube, organic nanoclay, or an organoclay. Foranother example, the nanocrystalline filler may be montmorillonite,bentonite, kaolinite, hectorite, halloysite, etc.

In some embodiments, the sol-gel polymer composites comprise areinforcing agent such as an inorganic filler, e.g., silicon dioxide(SiO₂).

In some embodiments, the sol-gel polymer composites further comprisecalcium phosphate as the reinforcing agent. In other embodiments, thesol-gel polymer composites form a double network hydrogel forreinforcement of a strong solid phase. Double network gels arecharacterized by a special network structure consisting of two types ofpolymer components and have both a high water content (about 90% w/w)and high mechanical strength and toughness.

The composition may also optionally include one or more pharmaceuticalagents, particularly antimicrobial agents having antibacterial,antiviral, anti-mycotic or anti-parasitic activity and the like. Thepharmaceutical agent or agents will become trapped in the compositionupon its formation and be released from the composition immediately orover a period of time.

Since the typical offending pathogen in mastitis is bacterium, thesol-gel polymer composites may desirably contain the antibacterialagent. There are a variety of antibacterial agents available for use inanimals. These antibacterial agents include, but are not limited to, thefollowing: macrolides, for example, tulathromycin (Draxxin®),tildipirosin (Zuprevo®), tilmicosin (Micotil®), tylosin phosphate(Tylan®), and gamithromycin (Zactran®); cephalosporins, for example,ceftiofur sodium (e.g., Naxcel® and Excenel®), ceftiofur hydrochloride(e.g., Excenel RTU®, Excenel RTU EZ®, Spectramast®), ceftiofurcrystalline free acid (Excede®), cefovecin sodium (Convenia®), andcefpodoxime proxetil (Simplicef®); lincosaminide antibiotics, forexample, lincomycin (Lincomix®), pirlimycin hydrochloride (Pirsue®), andclindamycin hydrochloride (Antirobe®); fluoroquinolones, for example,danofloxacin (Advocin®), enrofloxacin (Baytril®), and marbofloxacin(Zeniquin®); and tetracyclines, for example, chlortetracycline,oxytetracycline, and doxycycline. Other antibacterial agents include,but are not limited to, a penicillin derivative such as amoxicillintrihydrate alone or with clavulonic acid (Clavamox®), spectinomycin(Adspec®), potentiated sulfonamides including trimethoprim/sulfadiazine(Tucoprim®) and sulfadimethoxine/ormetoprim (Primor®); chloramphenicoland its derivatives such as thiamphenicol and fluorinated syntheticanalogs of thiamphenicol such as florfenicol (for example, Nuflor® andNuflor® Gold). An antimicrobial agent may be administered simultaneouslyor sequentially with the compositions of the present disclosure.

The sol-gel polymer composites of the disclosure can form durable sealsor strong solids in response to one or more physiological stimulus,typically at a temperature of about 37° C. Ideally, a “strong” solidsol-gel polymer means that the elastic modulus G′ (also referred to asthe storage modulus (G′)) is at least about 420 Pa or higher atphysiological temperature. The “strong solid” is generally a gel orsolid formed after the sol-gel phase transition for which G′ atphysiological conditions (e.g., 37° C., and/or near physiological pH) isgenerally above about 560 Pa, often about 600 Pa or higher, but alsoembracing from about 450 Pa to about 10,000 Pa and including, but notlimited to, values of about 490 Pa, about 560 Pa, about 650 Pa, about800 Pa, about 1700 Pa, about 1900 Pa, about 2500 Pa, about 5500 Pa about6000 Pa, about 6500 Pa, about 7000 Pa, about 7500 Pa, about 8000 Pa,about 8500 Pa, about 9800, about 9000 Pa, about 9500 Pa, about 10,000 Paor higher, and the like. In some embodiments, G′ is from about 450 Pa toabout 600 Pa, about 500 Pa to about 1000 Pa, about 1000 Pa to about 6000Pa, about 5000 Pa to about 9800 Pa, about 7000 Pa to about 10,000 Pa,from about 8500 Pa to about 10,000 Pa, etc. The “strong” solid sol-gelpolymer composite of the disclosure is generally stronger than knownchitosan hydrogels, which are known to be weak (in other words, a strongsolid sol-gel polymer composite has stronger or higher mechanicalproperties than known chitosan hydrogels). In other embodiments, the G′of the sol-gel polymer composite useful as a teat sealant may be fromabout 420 Pa to 9,800 Pa or above, wherein the polymer composite hasbeen unexpectedly found to be infusible without leakage and to form adurable seal in reaction to physiological stimuli similar to thestronger polymer composites described herein.

Advantageously, the sol-gel polymer composites possess thermalthickening properties making them capable of gelling or solidifyingquickly in response to one or more physiological stimulus, such asphysiological temperatures, to form a long-lasting seal or a strongsolid without the addition of any other agents. Moreover, the sol-gelpolymer composites gel or solidify very rapidly in response to one ormore physiological stimulus and form the seal or solid mass havingmechanical or viscoelastic properties as discussed herein, wherein thesolid possesses sufficient strength to uniquely enable it to withstandmechanical or hydraulic pressures under physiological conditions in theanimal. In some instances, the sol-gel polymer composites may gel orsolidify in seconds, i.e., instantly or almost instantly, after exposureto the physiological stimulus, for example, after infusion into a dairyanimal. The sol-gel polymeric composites provided herein undergo aliquid-solid phase transition so fast in response to physiologicalstimuli that a plug is rapidly formed at the site of injection. Thesol-gel polymer composites show favorable shear-thinning properties,i.e., their viscosity will decrease upon increasing shear rate, whichbeneficially allow the sol-gel polymer composites to be capable of beingeasily infused or, before infusion, deformed in a syringe at roomtemperature, even if solidification has already occurred. Due to thebeneficial shear-thinning properties, the sol-gel polymer composites canembrace solid structures having high porosity and/or elasticity forbetter manipulation of the material to seal the teat area yet to permitthe release of pharmaceutical agents required for treatment of mastitis.

In contrast to the disadvantages of using weak gels known in the artthat tend to spread and to leak in dynamic physiological environments,it is a further benefit of the present sol-gel polymer composite in itsability to solidify rapidly such that the composite can provide aninstant-gelling, resilient seal or strong solid that permits easyinfusion through a syringe without leaking or dripping and rapidformation of a no-leak, no-drip sol-gel plug after infusion into theteat canal or teat sinus of a dairy animal. In some instances, sol-gelpolymer composites are capable of being administered with asingle-barrel syringe.

Due to the properties of the sol-gel polymer formulations to respond tochanges in temperature, pH and ionic strength, they can formlong-lasting seals or strong gels/solids when no force is applied, butthey can flow and are syringeable upon application of external force,e.g., in a syringe. The composites can also form a durable, elastic gel,foam or porous solid after infusion.

Other valuable technical effects that are seen in the specially designedsol-gel polymer composites are the ability to form hydrophobicsubstitution in the polymers to increase viscosity, microgel spherescapable of crosslinking in physiological fluids and microgel spherescapable of being used for drug release as well as to control the rate ofdegradation in an animal and to form a porous solid with a particularpore size in a subject and desirable viscoelastic properties atphysiological temperature.

Beneficially, the water-based sol-gel polymer formulations are capableof being infused directly into the teat canal of the milk-producinganimal and form a firm sealant during the dry period. The formulationscan create this impermeable seal at 37° C. in the presence of milk andunder high ionic content that is usually seen upon the drying off of themammary gland.

Method for Making Sol-Gel Polymer Composites

Sol-gel processes are wet-chemical techniques widely used in the fieldof materials science and engineering. Such methods are used primarilyfor the fabrication of materials starting from a colloidal solution(sol) that acts as the precursor for an integrated network (or gel) ofdiscrete particles or network polymers. In a sol-gel process, a fluidsuspension of a colloidal solid (sol) gradually evolves towards theformation of a gel-like diphasic system containing both a liquid phaseand a solid phase whose morphologies range from discrete particles tocontinuous polymer networks (for general information, see C. J. Brinkerand G. W. Scherer, 1990, Sol-Gel Science: The Physics and Chemistry ofSol-Gel Processing, Academic Press, ISBN 0121349705; L. L. Hench and J.K. West, 1990, The Sol-Gel Process, Chemical Reviews 90:33).

In some instances, a reactivity and a functionality of a polymer can bealtered by addition of a set of functional groups including, but notlimited to, an acid anhydride, an amino or salt, an N-substituted amino,an amide, a carbonyl, a carboxy or salt, a cyclohexyl epoxy, an epoxy,glycidyl, hydroxy, an isocyanate, urea, an aldehyde, an ester, an ether,an alkyl, an alkenyl, an alkynyl, a thiol, a disulfide, a silyl or asilane, or groups selected from glyoxals, aziridines, active methylenecompounds or other β-dicarbonyl compounds (e.g., 2,4-pentandione,malonic acid, acetylacetone, ethylacetone acetate, malonamide,acetoacetamide and its methyl analogues, ethyl acetoacetate, andisopropyl acetoacetate), a halo, a hydride, or other polar or H bondinggroups and combinations thereof. Such functional groups can be added atvarious places along the polymer, such as randomly or regularlydispersed along the polymer, at the ends of the polymer, on the side,end or any position on the crystallizable side chains, attached asseparate dangling side groups of the polymer, or attached directly to abackbone of the polymer. Also, a polymer can be capable ofcross-linking, entanglement, or hydrogen bonding in order to increaseits mechanical strength or its resistance to degradation under ambientor processing conditions.

As can be appreciated, a polymer can be provided in a variety of formshaving different molecular weights, since a molecular weight (MW) of thepolymer can be dependent upon processing conditions used for forming thepolymer. Accordingly, a polymer can be referred to herein as having aspecific molecular weight or a range of molecular weights.

The sol-gel polymer composites rely on fast, salt-induced andthermoreversible gelling systems based on chitosan that are formed bymixing chitosan with hydrophilic polymers (e.g., water-solublepolysaccharides) that create highly viscous thermosensitive gels. Thesewater-swellable polymer composite formulations undergo a rapid gelationupon increasing temperature, pH and ionic strength. The compositestypically contain two hydrophilic polymers and ionic gelators. The firstpolymer undergoes temperature-induced gelation and enables formation ofan elastic gel in the teat canal at physiological temperatures, e.g., atabout 37° C. The second polymer forms a gel upon contact with ionicgelators (gelation agents) introduced to the formulation. With theaddition of the gelation agent (i.e., a thermogelling element), thesystem creates physical crosslinking. The gelation is basically due tophysical conformational changes in the polymers that are the majoringredients in the product, with no covalent crosslinking bonds betweenpolymers being formed. The strength of the gel of the second polymerdepends on the amount of gelator added, as well as pH and ionic strengthof the formulation, which typically has a pH of about 5.1 to 6.8 and anionic strength of about 5 g/L. The two hydrophilic polymers have aprofound effect on the gelation of chitosan, leading to a fast responseto stimuli such as salt addition and body temperature. As a result, thegels are reinforced upon changes of pH and ionic content in the dryingteat canal.

By carefully adjusting polymer, salt, and gelation agent concentrationsin the system, it is possible to fine-tune the gelation temperature andthe mechanical properties or integrity of the system, both below andabove the gelation threshold, including the speed and reversibility ofgel formation under physiological conditions (e.g., temperature), aswell as the biocompatibility, the viscoelastic properties (e.g., G′),the permeability/porosity of the system and the durability of the sealor strong solid that is formed after gelation. For example, the polymer,salt addition and the gelation agent can be manipulated to make thesol-gel polymeric composites suitable for specially designed human orveterinary uses which require a macroporous solid with a specific poresize and having durable, elastic properties, or in applications thatrequire structures that are solid with a high porosity or with aparticular pore size to allow free passage of biomolecules such asantibiotics.

Moreover, the sol-gel polymer formulations can be preferentiallydesigned for compatibility and use with cheese starter cultures, forexample, Lactococcus lactis, L. lactis subsp. cremoris, Streptococcusthermophilus, and the like. Since the live starter culture needs toachieve proper acidification for the process of making cheese to work,the polymer formulations' acidity can be suitably adjusted by alteringthe salt component to avoid interfering with culture activity andgrowth. Thus, to make the sol-gel polymer formulations beneficiallycompatible with cheese starter cultures, the salt component is easilyvaried to neutralize the acidity of the final product. Typically, forinstance, the formulation at a pH of approximately 6.8 (relativelyneutral), does not inhibit standard bacterial cultures and would finduse with cheese starter cultures.

To illustrate certain formulations of this disclosure, the sol-gelpolymer composite comprises about 7.3% w/w methyl cellulose, about 1.8%w/w chitosan, about 9.4% w/w sodium pyrophosphate tetrabasic (assolution salt), about 0.05% w/w sodium pyrophosphate tetrabasic (assolid salt), and about 82% w/w 0.1M aqueous acetic acid. This compositeis referred to herein as the “F1” or “F1-20141210” formulation. In asecond embodiment, the sol-gel polymer composite comprises about 7.3%w/w methyl cellulose, about 1.8% w/w chitosan, about 9.1% w/wβ-Glycerophosphate disodium (as solution salt), about 3.0% w/wβ-Glycerophosphate disodium (as solid salt), and about 82% w/w 0.1Maqueous acetic acid. This composite is referred to herein as the “F4” or“F4-20141210” formulation. For comparison, other sol-gel polymercomposites were prepared, such as the F2 or the F3 formulations shown inthe below Tables B and C. In further instances, the sol-gel polymercomposite comprises about 16% w/w, 17% w/w or 18% w/w Pluronic® F127 ina 0.5% w/w, 1% w/w, or 2% w/w chitosan solution in acetic acid. Inanother embodiment, the sol-gel polymer composite comprises one of thechitosan-Pluronic® F127 solutions set forth in the below Table 4.

As a general rule, the amounts of the ingredients in the sol-gel polymercomposite formulations of the present disclosure may vary somewhat. Inthe above illustration of the F1 to F4 formulations, for example, theamount of the methyl cellulose may range between about 4% w/w to about12% w/w, the chitosan may range between about 0.5% w/w to about 4% w/w,the solution salt or gelling agent may range between about 6% w/w toabout 12% w/w, the solid salt may range between about 0.01% w/w to about4% w/w, and higher concentrations than 0.1M of the aqueous acetic acidmay be used. It should nevertheless be appreciated that the ranges ofcertain combinations may be readily adjusted, including higher or loweramounts than the stated ranges, in order to form a gel or a hydrogelhaving the desired properties described herein.

Uses of Sol-Gel Polymer Composites

The sol-gel polymeric composites provided herein undergo a liquid-solidphase transition in response to one or more physiological stimulus. Theymay have use therefore in a wide range of animal and human healthapplications where it is desirable to inject a liquid that solidifiesrapidly after injection in a subject. In particular, sol-gel polymercomposites may have use in applications where it is desirable to beprovided with solid structures that have high porosity, elasticity orsufficient strength to withstand mechanical or hydraulic pressures underphysiological conditions in a subject.

It should be understood that suitability of sol-gel polymer compositesfor a particular application will be dictated by numerous factors, suchas their biocompatibility, mechanical integrity, speed and reversibilityof gel formation under physiological conditions (e.g., temperature),mechanical or viscoelastic properties (e.g., G′), porosity/permeability,and durability. In accordance with the present technology, suchproperties can be determined by adjusting polymer and gelation agentconcentrations in the system, as described herein. For example, in someembodiments sol-gel polymer composites are suitable for use inapplications that require a macroporous solid with a specific pore sizeand having durable, elastic properties, or in applications that requirestructures that are solid with a high porosity or particular pore sizeto allow free passage of biomolecules such as glucose, oxygen, orinsulin.

In some embodiments, sol-gel polymeric composites are suitable for usein applications that require formation of no-leak, no-drip plugs insideorifices in a subject. For example, they would find use as mucoadhesiveimplants, ocular drops, transdermal patches, dental implants, vaginalsuppositories, etc., which need no-leak, no-drip plugs, as do artificialspinal disks and cartilage.

For other embodiments, the sol-gel polymeric composites are suitable foruse in tissue engineering where the implant can replace deteriorated orotherwise damaged cartilage within a joint. In this regard, thecomposites are suitable for use as artificial cartilage. Since cartilagetissue is important for normal joint function, there is a need forartificial cartilage for therapeutic uses to replace tissue damaged frominjury or aging. Potential materials for use in artificial cartilageneed to be viscoelastic, strong, and durable, like the sol-gel polymericcomposites provided herein. Sol-gel polymeric composites may, therefore,be used for joint surgery, implanted in a knee joint, used as cornearepair material, or used for repairing, replacing, or therapeuticallytreating tissues and body parts. Sol-gel polymeric composites may form adurable, elastic gel after injection and in the presence of ions in thesynovial fluid and bone, forming an artificial cartilage, meniscus ornucleus pulposus.

The composites are also beneficially useful as injectable implants totreat osteoarthritis, rheumatoid arthritis, other inflammatory diseases,generalized joint pain or other joint diseases, for wound healing, or assuppositories. In some embodiments, sol-gel polymeric composites may beinjected to rapidly form a plug in a subject, particularly where theliquid-solid transition occurs so quickly in response to physiologicalstimuli that a plug is instantly formed at the site of injection.Sol-gel polymeric composites may, for example, be injected into Isletsof Langerhans in the pancreas in order to immune-isolate the islet cellsand allow free passage of glucose and oxygen.

In certain embodiments, sol-gel polymeric composites are useful asembolic or occlusion agents, e.g., to block arteries or to starve cancercells. In other embodiments, sol-gel polymeric composites are useful asultrasound contrast agents. In yet other embodiments, sol-gel polymericcomposites are suitable for injection along with cells that can form amacroporous or microporous substrate, for tissue engineering.

Alternative embodiments find that the sol-gel polymeric composites aresuitable for use as a bulking agent for reconstructive or cosmeticsurgery, for drug delivery systems, e.g., as a platform for slow-releasedelivery of therapeutic agents, or for treatment of varicose veins,e.g., forming an injectable foam. In some embodiments, sol-gel polymericcomposites are suitable for use as bulking agents to treat uterinefibroids or as dental implants. The composites are further suitable foruse in staunching wounds, e.g., forming a porous solid after injectionthat serves to block blood flow e.g. to block dental tubules, and asbrain implants, and as film-forming polymers on teeth.

Sol-Gel Polymer Composites as Teat Sealants for Animals

It should be understood that desired characteristics of the teat sealantencompassing the sol-gel polymer composites of the present disclosurewill vary depending upon the intended usage of the sealant, such aswhere it will be applied (e.g., exterior mammary tissue or within theteat cavity), the formulation of the sealant and other factors. However,some general characteristics of the teat sealant can be stated. Wherethe sealant is placed intramammary in the teat canal, for instance, thetailorable shear thinning characteristic allows for ease of infusion aswell as removal by manual stripping from the teat canal. It isadvantageous that the adhered teat sealant is easily removable from theteat. By adjusting the formulation of the teat sealant, the sol-gelpolymer composites can be made strong enough such that the sealant canbe readily peeled off the teat and removed in one cohesive unit in thefirst strip leaving little to no material behind. When the sol-gelpolymer composites are used as plugs at the macro or micro level, theycan be formulated to embrace an appropriate range of adhesion whichallows the in situ formed compositions to adhere to tissue and to stayin place as long as necessary while still being easily removed manuallyfrom the site. In addition, if these compositions are plugged in alocation where a barrier to retain or absorb fluid is necessary, thecomposition can be formulated to absorb the fluid naturally whileremaining in their desired location.

It is further advantageous that the in situ formed teat sealant of thepresent disclosure is conformable to the shape of the teat or teatcanal. The pliability property of the sol-gel polymer composite allowsthe sealant to conform to the topography of the teat surface or canal aswell as the tissue surface around the teat for a good fit. Suchconformability also extends the longevity, comfort and efficacy of theteat sealant.

The sol-gel polymeric composite useful as a teat sealant in thedisclosure is also safe and stable. The nontoxic property of the sealantprovides safety to the target animal as well as the human handler of theanimal and applier of the sealant on the animal. Because the teatsealant causes no residual accumulation of sealant in milk lines orrelated parlor equipment, the nontoxicity of the sealant also ensuresthat human food products, such as the milk and milk products made fromthe milk obtained from the treated animals, are safe to consume. All ofthe composition ingredients are non-heavy metal in nature,biocompatible, biodegradable tissue-adherent and nonirritating to theanimal in the amounts present in the final teat sealant formulations.Since the sol-gel polymer composites are suitable for infusion into themammary gland of the animal, they are made free of toxic materials,irritants, etc., and suitable for use under physiological conditions oftemperature, pH, ionic strength, etc. Moreover, the sol-gel polymercomposites can be readily sterilized by standard steam, dry sterilizers(autoclaves), gamma irradiation, electron beam methods (e-beamsterilization) and the like. Preservatives can also be included in thecomposition without altering the beneficial properties of the teatsealant. Depending on the delivery method, the viscosity is speciallydesigned in the formulation of the teat sealant to be made suitable fortopical application, infusion and the like. Thus, the viscosity of thesol-gel polymer composites is controlled so that the composition can besprayed or streamed onto or into the teat in such a way that anexcellent barrier is created. The sol-gel polymer composites are alsoreadily passed through a syringe and have excellent shear-thinningproperties which are necessary for molding the composite in a syringe atroom temperature for quick and easy product use by the animal handler.In particular, the sol-gel polymer composite is beneficially capable ofbeing infused using a single-barrel syringe. When infused directly intothe teat, the formulations have the ability to fill the teat canal andrapidly transition to a gel at body temperature. The shear thinningproperty of the formulations allows both ease of infusion andremovability.

Gelation of the composition on the teat is preferably rapid, to avoidrun off or loss of the composition from the place of application. Thegelling time can be about 5 minutes or less, preferably less than aboutthree minutes, more preferably less than about 30 seconds, and, in somesituations, as low as about 10 seconds or less, especially with externalapplication of the sol-gel polymer composites.

Several of the desired, aforementioned characteristics of the teatsealant useful in the present disclosure are obtained by adjustingpolymer and gelation agent concentrations in the sol-gel polymercomposite to modify the mechanical properties and permeability/porosityof the durable seal or strong solid. Basically, the adhesion and theswelling properties of the teat sealant are controlled by speciallydesigning the formulation. The sol-gel polymer composites, which possessa specific combination of adhesion and swelling properties, exhibitincomparable and new properties such as adhering to the animal's tissuefor an extended time period yet being easily removed as a teat barrierwhen the prophylactic treatment is finished, or being strong enough tobe peeled off the animal's teat in one piece or easily removed as asemi-solid or liquid yet being flexible enough to conform to theanimal's body for comfort and remain in place as an effective barrier toprevent or to reduce the incidence of mammary disorders.

Delivery of the Teat Sealant Compositions

Appropriate viscosity depends upon the delivery means to be employed.Generally, the composition should have a viscosity lower than about 800cps at room temperature or during conditions of use (that is, not in theanimal), preferably lower than 300 cps, more preferably lower than 200cps to be delivered via aerosol. Delivery through a pump spray normallyrequires a lower viscosity, such as less than about 150 cps. Spraywithout aerosol calls for a viscosity less than about 50 cps.

The teat sealant formulation is applied to the teat by conventionalmeans such as, for example, a spray or stream from a syringe, pump,spray nozzle, aerosol, dip, or other type of device. A combination ofthe spray and stream may be applied in a method similar to a showerhead, whereby multiple streams provide the simulated broad coverage of aspray application. The sol-gel polymer composites are sprayed orstreamed externally onto the teat tissue whereupon they form a barrierseal.

For application by infusion inside the teat, as in the teat sinus orcistern, any veterinary syringe having a tapered syringe end, a teatneedle or intramammary tip made especially for insertion of solutions into the teat canal may be used. For an example, the gels can be insertedthrough a conventional infusion cannula or infusion nozzle using astandard 5 or 6 mL syringe. An effective amount of the teat sealant thatwill form the desired physical barrier in a teat canal in order toprevent or treat a mammary disorder depends upon the dairy animalspecies and size of its teats. Typically, a volume of between 2 and 3 mLis satisfactory to adequately fill the teat canal but the amount mayvary and can be easily titrated by the handler infusing the sealant intothe teat.

Generally, about 0.5 to 5.0 mL of the composition will be administeredto an animal teat, preferably about 1.0 to 4.0 mL, more preferably about2.0 mL or higher, and even more preferably about 3.0 mL. Formulationsmay be pre-loaded into syringes for easy unit dose administration.Desirably, the composition is administered when the dairy animal entersinto the dry period at the end of the lactation cycle or during the dryperiod of the animal, especially when it is a heifer or cow.

The composition may also be delivered externally or topically to theteat from a spray device or a stream device. The spray device mayinclude a container having a dispenser for spray delivery of the liquidcomposition. The type of container used is variable, depending uponcompatibility with the composition and the spray dispenser and can beglass, plastic, or metal. If the solutions are of a low enoughviscosity, a spray delivery may be achieved with simple mechanicalforces such as those achieved when depressing the plunger of a syringeby hand through an appropriately designed nozzle. It may be desirable toapply several layers of the composition to the teat to ensure adequatecoverage of the teat. In any case, an effective amount for forming thephysical barrier can be readily determined by visual appearance of thesealant on the teat.

The composition can also be delivered using a syringe outfitted with aspray head. Generally, any chemical, mechanical or electronic method forpropelling the liquid composition as a spray from the container isappropriate. In one aspect, a compatible liquid or gaseous aerosolpropellant is placed in an appropriate container along with thecomposition and the dispenser includes a valve mechanism that enablesatomized spray delivery of the liquid composition. Desirably, anintramammary infusion device may be used to deliver the teat sealantcomposition directly to the teat. The device can have a singledispenser, such as a spray tip from Nordson Corporation (Westlake, Ohio,U.S.A.). The device may include a meter so that the quantity ofcomposition can be controlled.

Examples of devices that could be used, or modified for use, to deliverthe compositions as teat sealants include those described in WO2015/038281 (Zoetis), U.S. Patent Application No. 2015/0080841 (C. M.Bradley et al.), U.S. Pat. No. 5,989,215 (Y. Delmotte et al.), U.S. Pat.No. 8,353,877 (S. Hallahan et al.), WO 2003/022245 (Bimeda Research &Development Limited), and WO 2013/021186 (Norbrook LaboratoriesLimited).

The foregoing description shows how to make the new sol-gel polymercomposite formulations as well as their unique properties for use in thepresent disclosure. The following examples demonstrate other aspects ofthe disclosure. However, it is to be understood that these examples arefor illustration only and do not purport to be wholly definitive as toconditions and scope of this disclosure. Chemicals were purchased fromSigma-Aldrich in St. Louis, Mo. unless indicated otherwise. It should beappreciated that the sequence of steps in the preparation of the sol-gelpolymer composites is not critical and may be varied from the examples.For instance, the order in which the ingredients are introduced into atank can be altered (such as adding the methyl cellulose first insteadof adding chitosan first and the like) without detriment to the finalproduct. It should be further appreciated that when typical reactionconditions (e.g., temperature, reaction times, etc.) have been given,the conditions both above and below the specified ranges can also beused, though generally less conveniently. The examples are conducted atroom temperature (about 23° C. to about 28° C.) and at atmosphericpressure. All parts and percentages referred to herein are on a weightbasis and all temperatures are expressed in degrees centigrade unlessotherwise specified.

A further understanding of the disclosure may be obtained from thenon-limiting examples that follow below.

Example 1 Preparation and Properties of Sol-Gel Polymer Composites

Four polymer composite formulations (F1, F2, F3, and F4) were preparedas follows below. Tables A, B, C, and D show the F1, F2, F3, and F4formulations respectively. Formulations were prepared in batches ofabout 30 g, 110 g, or 165 g, as indicated in the tables.

TABLE A F1 Formulation Weight chitosan/gelator Substance (g) Solid (g) %Solid (w/w) Comments methyl 12 12.000 7.251% 38.70967742 viscous clearliquid, cellulose pH 6, viscosity chitosan 3.0 3.000 1.813% decreasedupon sodium 15.5 0.078 0.047% autoclaving pyrophosphate tetrabasic (as 5g/L solution) 0.1M aq. acetic 135.0 acid Total 165.5 15.1 9.110% methyl8 8.000 7.271% 40 viscous clear gel, pH 6 cellulose chitosan 2.0 2.0001.818% sodium 10.0 0.050 0.045% pyrophosphate tetrabasic (as 5 g/Lsolution) 0.1M aq. acetic 90.0 acid Total 110.0 10.1 9.157% methyl 2.42.400 8.000% 40 viscous clear liquid, cellulose pH 6, viscosity chitosan0.6 0.600 2.000% decreased upon sodium 3.0 0.015 0.050% autoclavingpyrophosphate tetrabasic (as 5 g/L solution) 0.1M aq. acetic 24.0 acidTotal 30.0 3.0 10.050%

TABLE B F2 Formulation Weight chitosan/gelator Substance (g) Solid (g) %Solid (w/w) Comments methyl 2.4 2.400 8.000% 2 white elastic paste,cellulose pH 7, viscosity chitosan 0.6 0.600 2.000% decreased uponpotassium 3.0 0.300 1.000% autoclaving phosphate dibasic trihydrate (as100 g/L solution) 0.1M aq. 24.0 acetic acid Total 30.0 3.3 11.000%methyl 8 8.000 7.271% 2 white elastic paste, cellulose pH 7, viscositychitosan 2.0 2.000 1.818% decreased upon potassium 10.0 1.000 0.909%autoclaving phosphate dibasic trihydrate (as 100 g/L solution) 0.1M aq.90.0 acetic acid Total 110.0 11.0 10.020%

TABLE C F3 Formulation Weight Solid chitosan/gelator Substance (g) (g) %Solid (w/w) Comments methyl cellulose 2.4 2.400 8.000% 3.8 Opaque gel,pH 7, chitosan 0.6 0.600 2.000% viscosity sodium 3.0 0.158 0.525%decreased upon pyrophosphate autoclaving tetrabasic/potassium phosphatedibasic trihydrate (as 2.5 g/L and 50 g/L solution) 0.1M aq. acetic acid4.0 Total 30.0 3.2 10.525% methyl cellulose 8 8.000 7.271% 2 whiteelastic paste, chitosan 2.0 2.000 1.818% pH 7, viscosity sodium 10.00.525 0.477% decreased upon pyrophosphate autoclavingtetrabasic/potassium phosphate dibasic trihydrate (as 2.5 g/L and 50 g/Lsolution) 0.1M aq. acetic acid 90.0 Total 110.0 10.6 9.589%

TABLE D F4 Formulation Weight Solid chitosan/gelator Substance (g) (g) %Solid (w/w) Comments methyl cellulose 12 12.000 7.273% 0.6 Opaque gel,pH 7, Chitosan 3.0 3.000 1.818% phase separation β- 15.0 4.884 2.960%may occur during Glycerophosphate sterilization disodium (as 44% aqueoussolution) 0.1M aq. acetic 135.0 acid Total 165.0 19.9 12.051% methylcellulose 2.4 2.400 8.000% 0.6 Opaque gel, pH 7, chitosan 0.6 0.6002.000% phase separation β- 3.0 0.977 3.256% may occur duringGlycerophosphate sterilization disodium (as 44% aqueous solution) 0.1Maq. acetic 24.0 acid Total 30.0 4.0 13.256% methyl cellulose 8 8.0007.271% 0.6 Opaque gel, pH 7 chitosan 2.0 2.000 1.818% β- 10.0 3.2562.959% Glycerophosphate disodium (as 44% aqueous solution) 0.1M aq.acetic 90.0 acid Total 110.0 13.3 12.071%

The F1 and F4 formulations were prepared as follows: First, 0.1M AcOHwas prepared by adding 0.813 g (0.772 mL) of AcOH to 135.5 mL of waterfor F1. For F4, 0.81 g (0.77 mL) of AcOH was added to 135.0 mL of water.Next, a solution of methyl cellulose (12 g), chitosan (3 g) in 0.1N(0.1M) AcOH (135.5 mL for F1 and 135.0 mL for F4) was prepared. Thesolution was prepared by addition of polymer powders to 0.1N (0.1M) AcOHheated to 85-90° C. with vigorous stirring. Heating was continued untila homogeneous dispersion was obtained (about 5 min.). The solution wasthen cooled to room temperature with stirring (300 rpm). Upon cooling,the solution clarified and became very viscous, necessitating reductionof stirring speed to 60-100 rpm. Cooling and clarification took about 1hour. The solution was then stored at 4° C. until future use.

Solution of gelling agent (salt works in formulation as gelation agent,also referred to herein as “gelator” agent) in water was then preparedby mixing salt in powdered form with water. The concentrations for saltsolutions were as follows: For F1, sodium pyrophosphate tetrabasic (5g/L) was prepared by adding 100 mg of sodium pyrophosphate tetrabasicinto 20 mL of water and stirring it until completely dissolved. For F4,β-glycerophosphate disodium hydrate (as 44% w/w aqueous solution) (44 gper 66 g of water) was prepared by adding 22 g of β-glycerophosphatedisodium hydrate (as 44% w/w aqueous solution) into 33 mL of water andheated at 45° C. with stirring until completely dissolved.

Gelator solution thus prepared was added to cold polymer solution withslow mechanic stirring (with a mixer at 100 rpm) until homogeneous.Significant viscosity enhancement and bubble formation was observed. Thesolution was then left to rest overnight at 4° C. Formulations weresterilized by autoclaving on the liquid cycle.

The F2 and F3 formulations were prepared as follows: 0.1M AcOH wasprepared by adding 0.6 g (0.57 mL) of AcOH to 100 mL of water. Asolution of methyl cellulose (8 g), chitosan (2 g), and sodium azide(0.025 g) was prepared in 0.1N AcOH (90 mL). The beaker with the stirrerbar was weighed. Solution was prepared by addition of powders (one shot)to 0.1N AcOH heated to boiling (85-90° C.) under stirring. Heating wasstopped and a homogeneous dispersion was obtained by stirring with aspatula for about 3 min. This was completed with addition of AcOH untilfinal weight (without salt) was achieved. The solution was then cooledto room temperature. Upon cooling, the solution clarified and becamevery viscous. Cooling and clarification took about 1 hour. The beakerwas covered with parafilm and stored at 4° C. for 24 hours (or untilfurther use).

A solution of gelling agent (salt as gelation agent or gelator) was thenprepared in water by mixing salt in powdered form with water to thedesired concentration. Gelation solution was added to the cold polymersolution under slow mechanical stirring (with a spatula or a mixer at100 rpm). A significant viscosity enhancement and bubble formation wasobserved. The solution was left to rest overnight at 4° C. The solutionwas centrifuged at 4000 rpm for 10 min. in order to eliminate airbubbles.

Several polymers were tested at various concentrations in theformulations to select most favorable polymers and concentrationsthereof for formulation performance. Various gelation agents were alsotested to tune the temperature responsiveness of the formulation, itssyringeability, and its flow properties. Four sol-gel polymer compositeformulations (F1-F4) having a broad range of flow properties were thenselected for further testing.

Preliminary characterization of the F1-F4 samples is shown in Table E.Note the pH of the gels was between about 6 and 7. In this experiment,it was shown that the gels could be injected through a standard 16-gaugeneedle using a 6 mL syringe, which demonstrates their unique ability tobe infused through a teat needle or nozzle for use as teat sealants. Forthe data in Table E, the formulations tested were those prepared usingthe ingredients and the amounts in Tables A-D for 110 g total.

TABLE E Properties of the Polymer Composite Formulations F1-F4 Plugformation Solid in the Formu- content Appearance Syringeability presenceof lation (%) pH (25° C.) (25° C.) milk (37° C.) F1 9% 6 Transparent + +gel F2 10% 7 Off-white gel + + F3 10% 7 Opaque gel + + F4 13% 7 Opaquegel + +

Plug formation in the presence of milk was tested by adding theformulations (1.5-2 mL) into test tubes containing 0.5 mL of milk (3.5%fat) at 37° C. All the formulations formed a gel plug on the surface ofmilk. The inspection of the interface between milk and gels showed thatwhile some degree of mixing between formulations and milk was expectedupon addition of the gel to the milk, the samples unexpectedly showedvery limited miscibility with milk upon formation of the plug. Theresults indicated that the formulations were able to form a gel in thepresence of milk and prevent leakage from the test tubes.

A stress sweep test (i.e., measurements of elastic and loss moduli as afunction of stress at fixed frequency) was performed to confirmshear-thinning character of the samples. The results are shown in FIG.6. For the data in FIG. 6, the formulations tested were those preparedusing the ingredients and the amounts in Tables A-D for 110 g total.

At low applied stresses, the values of elastic modulus G′ were constant.The elasticity of samples covered a broad spectrum, ranging from about10 Pa for F1 to about 440 Pa for F4. Shear-thinning behavior (i.e., adecrease of elastic modulus G′ as a function of the applied stress) wasobserved at higher stress values. The shear-thinning region started atlowest stress for the F2 formulation, followed by F1, F3, and F4. Theseresults confirm that the samples are syringeable and their infusion ispossible. Since the force applied during formulation infusion andpost-treatment teat stripping is well above 300 Pa, all formulationswould not undergo any delivery or recovery issues.

Next, the rheology of the formulations was monitored upon a sudden jumpin temperature from room to body temperature. The values of elastic andstorage moduli as a function of time at a constant stress of 1 Pa (i.e.,in the plateau region) and frequency (f=0.1 Hz) were followed. In thefirst step of the experiment (10 min.), the temperature was kept at 25°C. In the second step (10 min.), the temperature was fixed at 37° C. Theheating process between both steps took around 20 seconds. The resultsare shown in Table F and FIG. 7; they are expressed as elastic modulusG′ and loss tangent tan δ=G″/G′, the ratio of elastic and viscousproperties showing which one is dominant. With a tan δ value of 1, theelastic and viscous properties of the material are equal. The smallerthe loss tangent, the more elastic is the material. For physical gels,G′>G″ and tan δ<1. For viscous liquids, G″>G′ and tan δ>1. For the datain Table F and FIG. 7, the formulations tested were those prepared usingthe ingredients and the amounts in Tables A-D for 110 g total. It willbe understood that values can vary based on equipment and the rheologymethods employed; and, further, these values are relative from eachother depending on the particular testing protocols.

TABLE F Results of Rheological Tests for Sol-Gel Polymer CompositeFormulations F1-F4 (Values for G′ and tan δ were recorded 10 min. afterthe application of the stress. Angular frequency f = 0.1 Hz, oscillatorystress τ = 1 Pa) T = 25° C. T = 37° C. Formulation G′ (Pa) tan δ G′ (Pa)tan δ G′(37° C.)/G′(25° C.) F1 9.3 3.4 490 0.08 53 F2 71 0.26 1900 0.0627 F3 32 0.84 870 0.05 27 F4 420 0.13 8600 0.04 20

Elasticity of F1-F4 increased significantly with temperature, asevidenced by the values of the ratio of G′(37° C.)/G′(25° C.) (Table E)that changed from 53 (F1) to 20 (F4). At room temperature, F1 behavedlike viscous liquid (tan δ>1). Three other formulations showedsolid-like behavior that became more pronounced in the order F3<F2<F4.

At 37° C., elastic modulus increased significantly. This was accompaniedby a fast decrease of loss tangent, indicating the reinforcement of thegel structure. This process took about 1-2 min. for F2, F3 and F4. Itwas slightly longer (about 5 min.) for the F1 formulation.

In conclusion, four sol-gel polymer composite formulations covering abroad range of flow properties were prepared and characterized. Thepolymer composite formulations undergo a rapid gelation upon increasingtemperature, pH and/or ionic strength. The composites contain twohydrophilic polymers and ionic gelators. As described, the first polymerundergoes temperature-induced gelation and enables formation of anelastic gel at about 37° C. The second polymer forms a gel upon contactwith ionic gelators introduced to the formulation. The strength of thegel of the second polymer depended upon the amount of gelator added, aswell as pH and ionic strength of the formulation.

The samples were infusible due to their shear-thinning properties. Theyshowed temperature-induced thickening, i.e., their gel structure becamestronger upon an increase of temperature. Results indicated thatelasticity of the formulations, the onset of shear-thinning, the extentof temperature-induced thickening, as well as the time-scale of theseprocesses depend on the gelation agent used in the formulation,potentially allowing for precise tuning of flow properties.

Example 2 Effect of Sterilization on Sol-Gel Polymer CompositeFormulations

Autoclaving was tested as a method of sterilization. It is known thatpolymers similar to those used in the above formulations may undergosignificant degradation upon sterilization with ionizing radiation orethylene oxide. This degradation may be lessened by using hightemperature to sterilize the samples.

Sterilization was performed at 121° C. during 10 min. The total lengthof the cycle, including heating and cooling parts was about 45 min.Table 1 shows a comparison of the properties of the formulationsprepared with two different gelators (F1 and F4) before and aftersterilization. For the data in Table 1, the formulations tested wereprepared using the recipes in Tables A and D for 165.5 and 165 g total,respectively.

TABLE 1 Properties of Polymer Composite Formulations Before and AfterSterilization. Appearance Syringeability Formulation Conditions pH (25°C.) (25° C.) F1-20141210 Before 6 Transparent + sterilization slightlyyellowish gel After sterilization 6 Transparent + yellowish viscoussolution F4-20141210 Before 7 Opaque slightly + sterilization yellowishgel After sterilization 7 Opaque + yellowish gel

After sterilization, all the formulations could be injected through astandard 16-gauge needle using a 6 mL syringe, which shows theformulations can be injected as teat sealants through a teat needle ornozzle. The pH of the formulations did not change significantly uponsterilization. However, visual observation indicated that bothformulations showed a more pronounced yellow/brown color afterautoclaving and that their flow properties changed. In the case ofF1-20141210, there was a change from “gel” to “liquid” at 25° C.

Next, the changes in flow properties of the sterile and non-sterileformulations were monitored more closely upon a jump in temperature from25° C. to 37° C. The values of elastic and storage moduli as a functionof time were followed at a constant stress (1 Pa) and frequency (f=0.1Hz). In the first step of the experiment (10 min.), the temperature waskept at 25° C. In the second step (10 min.), the temperature was fixedat 37° C. The heating process between both steps took around 20 seconds.The results are shown in Table 2 and in FIG. 1, where they are expressedas elastic modulus G′ and loss tangent tan δ=G″/G′, the ratio of elasticand viscous properties showing which one is the dominant one. When thetan δ value is 1, the elastic and viscous properties of the material areequal. The smaller the loss tangent, the more elastic is the material.For physical gels, the values are G′>G″ and tan δ<1. For viscousliquids, the values are G″>G′ and tan δ>1. For the data in Table 2 andFIG. 1, the formulations tested were prepared using the recipes inTables A and D for 165.5 and 165 g total, respectively.

TABLE 2 Results of Rheological Tests for Polymer Composite FormulationsBefore and After Sterilization. Values for G′ and tan δ were recorded 10min after application of stress. Angular frequency f = 0.1 Hz,oscillatory stress τ = 1 Pa. T = 25° C. T = 37° C. FormulationConditions G′ (Pa) tan δ G′ (Pa) tan δ F1- Before sterilization 17 0.83560 0.07 20141210 After sterilization 8.7 0.91 650 0.05 F4- Beforesterilization 480 0.15 9800 0.03 20141210 After sterilization 440 0.105500 0.05

Sterile formulations were characterized by slightly smaller value ofelastic modulus compared to their non-sterile counterparts. Thisdifference was however small, especially in the case of F4-20141210. At37° C., elastic modulus increased significantly. This was accompanied bya fast decrease of loss tangent, indicating the reinforcement of the gelstructure. This process took about 1-2 min. for the formulationF4-20141210 and it was slightly longer (about 5 min.) for the sampleF1-20141210. Sterilization of the samples did not affect significantlythe kinetics of the viscosity enhancement nor the final values of G′ andtan δ reached by the samples at 37° C. (FIG. 1).

In conclusion, two composite gels were tested. The composites wereinfusible due to their shear-thinning properties. They showedtemperature-induced thickening, i.e., their gel structure becamestronger upon an increase of temperature. Although sterilization of thesamples induced a few subtle changes in the appearance of the samples,the elasticity of the formulations, the extent of temperature-inducedthickening, and the time-scale of this process are not affectedsignificantly by the sterilization process.

Example 3 Further Studies on Sterilization of Polymer CompositeFormulations

The F1 and F4 formulations are water-swellable polymer composites thatundergo a rapid gelation upon increasing temperature, pH and/or ionicstrength. The composites contain two hydrophilic polymers and ionicgelators. The first polymer undergoes temperature-induced gelation andenables formation of elastic gel at about 37° C. The second polymerforms a gel upon contact with ionic gelators introduced to theformulation. The strength of the gel of the second polymer depends onthe amount of gelator added, as well as pH and ionic strength of theformulation (typically, pH of about 5.1 to 6.8 and ionic strength ofabout 5 g/L).

In Example 2, it was shown that flow properties of the formulations werenot significantly affected by sterilization. This further study showsthe effect of sterilization on the water-swellable polymer composites.

For the sample F1-20141210, rheological measurements were repeatedtwelve weeks following sterilization. During this period the sample wasstored in the dark in closed plastic vials at room temperature. Visualobservation confirmed that the appearance and consistency of the sampledid not change significantly after 12 weeks of storage. In addition, novisual sign of microorganism growth was detected in the sample.

First, the changes in flow properties upon a jump in temperature from25° C. to 37° C. were monitored. The values of elastic and storagemoduli as a function of time at a constant stress (1 Pa) and frequency(f=0.1 Hz) were followed. In the first step of the experiment (10 min.),the temperature was kept at 25° C. In the second step (10 min.), thetemperature was fixed at 37° C. The heating process between both stepstook around 20 seconds. The results are shown in Table 3 and FIG. 2;they are expressed as elastic modulus G′ and loss tangent tan δ=G″/G′,the ratio of elastic and viscous properties showing which one is thedominant one. With a tan δ value of 1, the elastic and viscousproperties of the material are equal. The smaller the loss tangent, themore elastic is the material. For physical gels, the values are G′>G″and tan δ<1. For viscous liquids, the values are G″>G′ and tan δ>1. Forthe data in Table 3 and FIG. 2, the formulations tested were preparedusing the recipes in Tables A and D for 165.5 and 165 g total,respectively.

TABLE 3 Results of Rheological Tests for the F1-20141210 FormulationImmediately and 12 Weeks After Sterilization. Values for G′ and tan δwere recorded 10 min after the application of the stress. Angularfrequency f = 0.1 Hz, oscillatory stress τ = 1 Pa. T = 25° C. T = 37° C.Conditions G′ (Pa) tan δ G′ (Pa) tan δ Immediately after sterilization8.7 0.91 650 0.05 12 weeks after sterilization 10 1.3 420 0.05

The data show that storing the F1-20141210 sample for 12 weeks did notaffect significantly the flow properties or the extent of thethermothickening effect (FIG. 2). At 25° C., the sample showed losstangent of about 1. Heating to 37° C. was accompanied by a fast decreaseof tan δ, indicating the reinforcement of the gel structure. Thekinetics of this process was similar for freshly sterilized and 12week-old samples.

A stress sweep test (i.e., measurements of elastic and loss moduli as afunction of stress at a fixed frequency) was performed in order toconfirm the shear-thinning character of the sample stored for a 12-weekperiod. The results are shown in FIG. 3. At low applied stresses, thevalues of elastic modulus G′ were constant and similar to those obtainedfor the F1-20140825 sample (prepared using the recipe in Table A for 110g total) (i.e., non-sterile sample prepared under similar conditions tothose for F1-20141210, which was prepared in a batch of 165.5 g total).Shear-thinning behavior (i.e., a decrease of elastic modulus G′ as afunction of applied stress) was observed for both formulations at asimilar value of about 80 Pa. These results confirm that thesyringeability of the sample will not be significantly affected upon12-week storage at room temperature.

In conclusion, the physico-chemical stability of one of the compositegel samples was assessed after 12 weeks of storage at room temperature(in a closed container, in the dark). The results show that the flowproperties and syringeability of the sample are not significantlyaffected after 12 weeks of storage time.

Example 4 Preparation of Pluronic® F127 Gel Reinforced withNanocrystalline Cellulose

Development was performed in terms of (1) the onset of gelation offormulation at temperature close to body/skin temperature (32-35° C.),(2) syringeability of formulation at room temperature, and (3) timeneeded to induce the formation of the gel at 35° C.

All formulations were prepared using the following procedure: Pluronic®F127 polymer was molten in a 20 mL glass vial with heating and stirring(1.6 to 1.8 g). A filler (nanocrystalline cellulose (NCC)) was added, ifrequired (100 or 200 mg). Stirring and heating were continued for about5 min. Chitosan solution in 2% aq. AcOH was added under stirring to afinal weight of the sample of 10 g. The sample was kept under vigorousstirring overnight (no heating).

The onset of gelation was measured by heating the sample in a water bathto the desired temperature. For several formulations,syringeability/ability to flow was verified at room temperature bypassing about 3 mL of formulation from a 10 mL plastic syringe through a16-gauge needle.

In a separate experiment, gelation time was estimated by depositing adrop of the sample kept at room temperature through a 16-gauge needle onthe wall of a plastic Eppendorff tube heated to 37° C. The time neededto stop the flow of the solution was taken as an approximation of thegelation time.

Chitosan solutions were prepared as follows: A solution of chitosan (CH,2% w/w) was prepared by overnight vigorous stirring of chitosan powdersuspended in 2% AcOH, resulting in the formation of a transparent andhomogeneous yellowish solution. This solution was diluted to aconcentration of 1% and 0.5% with water followed by the addition of 0.5%NaOH solution to pH of about 6. Chitosan solutions are shown in Table 4.

TABLE 4 Chitosan Solutions Pluronic ® F127 final concen- GelationInjectability tration temper- at room (wt %) Solvent Additive aturetemperature 16% aq. AcOH (2%) N/A >40° C.  + 1% CH in AcOH N/A >40°C.  + 2% CH in AcOH N/A 38° C. + 17% aq. AcOH (2%) N/A 37° C. + 1% CH inAcOH N/A 37° C. + 2% CH in AcOH N/A 37° C. + 18% 0.5% CH in N/A 32° C. +AcOH 1% CH in AcOH N/A 31° C. +* 2% CH in AcOH N/A 31° C. +* 18% 1% CHin AcOH NCC 100 mg 29° C. + 17% 1% CH in AcOH NCC 100 mg 36° C. + NCC200 mg 34° C. + NCC 500 mg 29° C. + *For these samples, gelation timewas estimated; the droplet of formulation formed a gel almostimmediately after contact with the wall of the Eppendorf tube.

A graph showing dependence of the gelation temperature on the amount ofnanocrystalline cellulose (NCC) for the formulation of 17% Pluronic®F127 in 1% CH (pH at about 6) is shown in FIG. 4.

Gelation temperature of the formulations could be adjusted by adjustingthe concentration of Pluronic® F127. The presence of CH had no effect ongelation. All samples were syringeable at room temperature. Forformulations having 18% Pluronic® F127 in 1% or 2% CH solution, thegelation time was estimated to be almost immediate. Addition of NCC ledto a decrease in gelation temperature. NCC strengthened the gels andinduced temperature hysteresis (i.e., the samples become liquid uponcooling to temperatures lower than their gelation point as determinedupon heating).

Example 5 Synthesis of Modified Chitosan Gel

Carboxymethyl chitosan was prepared as follows: Carboxymethylation wascarried out by stirring chitosan (2 g) in 20% NaOH (w/v 100 mL) for 15min. Monochloroacetic acid (15 g) was then added dropwise to thereaction mixture and the reaction was continued for 2 hours at 40+/−2°C. with stirring. The reaction mixture was then neutralized with 10%acetic acid, and then poured into an excess of 70% methanol. Thecarboxymethyl chitosan produced was filtered using a G2 sintered funneland washed with methanol. The product was dried in a vacuum at 55° C.for 8 hours to give 6.5 g of dried carboxymethyl chitosan. The degree ofsubstitution of carboxymethyl chitosan (CMCh) was determined to be 0.75using methods as described (Biomacromolecules, Vol. 5, no. 2, 2004).

Polyvinyl acetate (PVA) (1 g) was dissolved in water (85 mL) at 45° C.After the PVA-water solution cooled to room temperature, acetone (15 mL)was added dropwise to the vigorously stirred PVA solution for 15 min toform about a 1% (w/v) PVA solution. Then the solution was kept at 5° C.for 24 hours until it became light yellow, which indicated that the longchains of PVA had shrunk to nanoparticles. Different amounts of CMCh(0.5, 1 and 2 wt %) were then added to the solution. The solution waspurged with N₂ for 30 min, then 4.0 mmol methylenebisacrylamide (MBA),0.4 mmol potassium persulfate (KPS), and 0.67 mmol tetramethylenediamine(TEMED) were added to the solution, and polymerization was carried outfor 15 hours at 30° C. The nanogels formed were either used directly orcould be frozen to form a freeze-dried powder which is easilypre-dispersed in water, forming nanoparticle dispersion, before use.

Acyl chitosan was prepared as follows: MeSO₃H was used as a solvent forchitosan in order to help protect the amino groups on the chitosanmolecules from acylation reaction. Typically, chitosan was dissolved inMeSO₃H at room temperature for 1 hour and octanoyl chloride was thenadded dropwise under stirring, with the molar ratio of octanoyl chlorideto the repeating unit of chitosan being equal to 0.66:1. The reactionwas allowed to continue for 5 hours at ambient temperature before it wasstopped by the addition of crushed ice. The resulting solution wasdialyzed for one day to remove most of the acid, and the remaining acidand ammonium salt were subsequently neutralized with NaHCO₃. The finalmixture was dialyzed against Milli-Q water for more than 3 days and thenlyophilized as acyl chitosan (AC) powder.

Example 6 Synthesis of PVA-Acylate

Materials: Polyvinyl alcohol (PVA), 186K, 87%-89% hydrolyzed: 10 g.R—COCl (e.g., lauroyl chloride, palmitoyl chloride, octanoyl chloride):1.68 g. Triethylamine (ET₃N): 2.25 mL. 1-methyl-2-pyrrolidone (NMP): 100g.

In alternate experiments, the materials were as follows: 10 g PVA, 186K, 87-89% hydrolyzed; 3.36 g R—COCl (lauroyl chloride, palmitoylchloride, or octanoyl chloride); 4.50 mL ET₃N; and 150 mL NMP.

Synthesis Procedure: PVA was added to hot NMP; if too viscous, then moreNMP was added, up to a final volume of 50 mL. After completedissolution, the R—COCl was added, followed by the ET₃N. The mixture wasleft at room temperature overnight with stirring. The PVA-acylate wasthen diluted by adding 3 times water, stirring, and then purifiedthrough dialysis over 5 days, and then lyophilized. Yield: 90%.

Viscosity at 6 s⁻¹ of PVA-acylate thus prepared is shown in FIG. 5.

Example 7 Evaluation of Retention and Tolerance of Teat Sealants Infusedin Cows

The objective of this study was to evaluate the retention and thetoleration of the novel use of two sol-gel polymer compositeformulations (F1 and F4, prepared using the formulations in Tables A andD, respectively, for 165.0 g total) as intramammary teat sealants (ITS)during the dry period of dairy cows.

Eight cows (adult lactating pregnant Holstein dairy cattle) were driedoff at initiation of study and each quarter was assigned one of twotreatment groups, T01 and T02. Group T01 received treatment with F1while Group T02 received treatment with F4. Spectramast® DC (ceftiofurhydrochloride) dry cow therapy was administered per quarter per labelinstructions prior to sealant infusion. Formulations were steamsterilized prior to infusion. All doses of the ITS were administered asintramammary infusions to all available quarters of an assigned cow. Alleight animals were allotted for consistent ultrasound evaluation atdefined times throughout the duration of the study. Approximately 60days after administration, upon calving, all test ITS was removed byhand stripping. General health observations and visual udder/quarterobservations were performed and recorded throughout the study.

The ITS formulations were delivered via either a syringe and specializedmixing tip attachment or ready-to-use plastets, intramammary (IMAM), 2.0mL. The partial insertion method of administration was used. F1 and F4were infused at 2.2 g+/−0.5 g. Both formulations were easily infusedalthough an initial resistance to initiate was noticed due to the shearthinning properties of the substrate. The F4 required more force toinfuse as compared to the F1. However, once flow was initiated, the F4became easier to infuse. All syringes had been autoclaved prior toinfusion. Infusion of the substrate into all test teats in animals waseasily accomplished. No syringes demonstrated defects. No syringesdemonstrated difficulty for infusion.

Calves were understood to be removed immediately at birth, and thereforenot allowed to suckle. Substrate was easily removed via manual strippingfrom all teats at the first milking of the animal post-calving,approximately 60 days after administration. No difficulty was indicatedupon removal of any of the substrate from the teats of any animals.

Samples of the first milk post-calving were collected to analyze forpresence of sealant substrate. Sample weights post removals were notdetermined due to the inability to distinguish teat sealant materialfrom colostrum. As a consequence of the teat sealants' properties ofbeing shear-thinning and temperature sensitive and because shear forcewas exerted on the formulations to remove them from the teat cistern,the formulation had thinned upon removal from the teat. Additionally,since these formulations gel at warmer temperatures and become liquid atcolder temperatures, placing the colostrum stripping samples collectedinto the refrigerator immediately post-collection further thinned theformulation. When the formulation returned to liquid form in the coldtemperature of the refrigerator, it became difficult by conventionalmeans to separate the sealant substrate from the colostrum to facilitateaccurate percent recovery for a measurement of total substrate removed.

A pathologist examined the interior of the teat canals for safety offormulations. No gross lesions were identified that had any relevance tothe test substrate or formulations. One incidence of one teat having amild subepithelial fibrosis and mononuclear infiltration was noted. Thisfinding could not be correlated to test substrate. All other teatsexamined were normal with no adverse findings. Tissues were sent forsectioning if any areas of gross pathologic concern were identified torule out any substrate concerns but none were identified. Microscopictissue assessment from this retention study resulted in nosubstrate-related findings.

The formulations had remained in the teats until calving, and werevisually assessed throughout the retention period via ultrasoundscoring. Ultrasound observations were performed by trained staff toassess presence of sealant on days 0, 1, 4 then weekly thereafter untilcalving. Numbers were logged on a visual scale from 0 (no sealantevident in teat cistern) to 5 (teat cistern appears fully blocked withsealant). Any unusual observations were recorded on the dailyobservations form. All formulations stayed in the teats throughout thedry cow period without incidence.

The results of the ultrasound scoring are shown in the below Tables 5and 6 for F1 and F2, respectively:

TABLE 5 F1 Formulation Ultrasound Scoring Cow ID Quarter Day 0 Day 1 Day4 Day 7 Day14 Day 20 Day 28 Day 47 556 LF 5   4.5 5 5 5 3.5 4 4 556 RR 55 5 4.5 5 4.5 3.5 3.5 557 LF 5 4 4.5 4 4 3.5 4 4 557 LR 4.5 4 4 4 4.53.5 3.5 4 558 LF 5 4 5 4.5 4 4 4 4 558 RF 5 4 5 4 4.5 3.5 3.5 4 559 LF4.5 4 4 4 4 4 3.5 4 559 LR 5 N/A 5 5 4 4 3.5 3.5 560 LR 4.5 4 4 4 4 4 43.5 560 RF 5 4 4 4 4 4 4 4 561 LR 5 4 3 3.5 5 3 3.5 3.5 561 RF 5 3 5 5 53.5 3.5 3 562 LR 4.5 4 3.5 3.5 3.5 3 3 3 562 RR 5 4 3 5 3.5 3.5 3.5 3.5563 LR 5 5 4 3 4 4 4 no video 563 RR 5 5 4 3 4 4 4 no video

TABLE 6 F4 Formulation Ultrasound Scoring Cow ID Quarter Day 0 Day 1 Day4 Day 7 Day 14 Day 20 Day 28 Day 47 556 LR 5 4 4.5 4 4.5 4 4 3.5 556 RF4.5 4 4.5 4 4.5 4.5 4 4 557 RF 4 4 4 4 N/A 4 3.5 4 557 RR 5 4 4 4 4 43.5 4 558 LR 5 5 4 5 4.5 3 3 4 558 RR 4.5 3.5 4 2.5 3.5 3.5 3.5 3.5 559RF 4.5 N/A 4 4 4 4 3 3.5 559 RR 4.5 4 4 5 4 3.5 3 3.5 560 LF 4 4 4 3 3.53.5 3 4 560 RR 5 3.5 4 3 3 4 3 4 561 LF 5 4 5 5 5 3.5 3.5 3 561 RR 5 3 4N/A 3.5 3.5 3 3.5 562 LF 4.5 4 5 4 3.5 3.5 3 4 562 RF 5 4 5 4 4 4 4 4563 LF 4.5 5 4 4 3.5 4 4.5 no video 563 RF 4.5 5 5 5 4.5 4.5 4.5 novideo

In sum, the study showed that the sol-gel polymer composite formulationswere easy to administer by intramammary infusions and to remove bymanual stripping. The results also indicated that the dairy cowstolerated the teat sealants without adverse side effects. Finally, theresults demonstrated retention of the teat sealants during the dryperiod of dairy cows until the sealants were physically removed at theend of the study.

Example 8 Evaluation of Retention and Tolerance of Teat Sealants Infusedin Cows

The objective of this study was to evaluate the retention and thetoleration of the novel use of two sol-gel polymer compositeformulations (F1 and F4, prepared using the formulations in Tables A andD, respectively, for 165.0 g total) as intramammary teat sealants (ITS)during the dry period of dairy cows.

Thirty cows (adult lactating pregnant Holstein dairy cattle) were driedoff at initiation of study and each quarter was assigned one of fourtreatment groups, T01 to T04. Two dose volumes of 2.0 and 3.0 mL performulation were infused to evaluate if volume had an effect onretention. Groups T01 and T02 received treatment with F1 at a volume ofabout 2.0 mL (actual delivered: average 1.99+/−0.19) and about 3.0 mL(actual delivered: average 2.88+/−0.15), respectively. Groups T03 andT04 received treatment with F4 at a volume of about 2.0 mL (actualdelivered: average 1.54+/−0.37) and about 3.0 mL (actual delivered:average 2.24+/−0.55), respectively. Animals were first acclimated to thefacilities, diet and water source for at least 10 days prior toinitiation of study. On study day −1+/−2 days, prior to morning milking,milk quarter samples were taken for somatic cell count analysis andbacterial assessment.

Spectramast® DC (ceftiofur hydrochloride) dry cow therapy wasadministered per quarter per label instructions prior to sealantinfusion. All doses of ITS were administered as intramammary infusion toall available quarters of an assigned cow. Weekly ultrasound evaluationof the teats of all thirty animals at defined times throughout the studywere completed in an effort to determine retention and evaluation of thesealants as a physical barrier.

The teat ends of all four quarters of fifteen animals were exposedweekly to a bacterial suspension throughout the dry period and afteradministration of teat sealant and dry cow therapy to simulate poorhygienic conditions in the dairy. A frozen stock of E. coli was used toprepare the bacterial suspension of 1×10⁶ colony forming units/mL(CFU/mL) in Trypticase Soy Broth. All four teat ends of the animals wereexposed to the E. coli preparation via a single dip from a dip cup onceweekly starting on day 7. Exposure to E. coli ceased post-calving orremoval of teat sealant.

Approximately 60 days after administration and at the first milkingpost-calving, test ITS was removed by hand stripping. Recovered sealantsamples were stored at room temperature. Beginning on the day of firstmilking post-calving, each cow was observed for clinical signs ofmastitis. Sterile quarter samples for bacteriological culture werecollected from each udder quarter after careful cleaning andpre-stripping of each individual teat on day 1, 2, 3, 7, 10 and 14post-calving. Milk and colostrum samples were collected at various timesthroughout the experiment in order to measure potential residues ormetabolites. Somatic cell counts and milk weight were also recorded toassess udder condition, presence of mastitis and quality of milk.

Ultrasound analysis for all substrates indicated the presence of asignificant amount of material throughout the dry period irrespective ofthe dose volume. Substrate was observed to undergo changes in appearancebetween days 14-35. Both formulations appeared to persist in the teatcanal and remained as a protective barrier throughout the dry perioduntil physical removal of the teat sealants. Post-calving substrate wasremoved from the teat through manual stripping.

Upon return to lactation, udder health, milk appearance and bacterialpresence were monitored over a two week period for all animals remainingin the study. While abnormal (elevated) udder health scores were notedin a small number of individual cows for all treatment groups over thecourse of the 14-day period, these could not be attributed to teatsealant failure. Neither udder health or milk quality scores werestatistically different between treatment groups.

Properly designed and monitored field studies will be needed to confirmprevention of mastitis. However, based on this retention study, it canbe concluded that the sol-gel polymer composite provides a sufficient,long-lasting physical barrier that is able to protect healthy dairyanimals from new infections or re-infections.

In summary, all treatments remained throughout the dry period andappeared to protect the teat through ultrasound evaluation. Allsubstrates were easily removable and no treatment related effects werenoted upon gross and microscopic evaluation of tissues. Overall, bothformulations F1 and F4 performed well with good retention throughout thedry period.

Example 9 Evaluation of Use of Sol-Gel Polymer Composite for DrugRelease

The objective of this study was to evaluate the release and theantimicrobial activity of an antibacterial agent via the sol-gel polymercomposite formulation. Formulation F2, prepared as describedhereinabove, was used and loaded with 20 mg of amoxicillin per gram offormulation without affecting its rheological property which byextension is related to plug formation for teat sealing.

Amoxicillin Loading:

A loaded F2 sol-gel polymer composite formulation was prepared bythoroughly mixing methylcellulose (8.0 m %), chitosan (2.0 m %),amoxicillin trihydrate (2.0 m %), and monopotassium phosphate (0.76 m %)into 0.1M acetic acid until an homogenous off-white cream was obtained.

The rheological data of the loaded F2 sol-gel was compared with that ofthe unloaded F2 sol-gel polymer composite (Table 7). The data from Table7 represent the average properties of several batches (from the same rawmaterials) of either unloaded (n=6) or loaded (n=3) sol-gels. The shearstorage modulus G′ and the shear loss modulus G″ values werestatistically equal between the two sol-gel formulations; the tan δvalues were thus trivially also equal. This demonstrates that theserelevant physical properties of the sol-gel polymer composite wereunaffected by the incorporation of 2 m % amoxicillin.

Amoxicillin Quantification:

In order to monitor amoxicillin release from the loaded sol-gel polymercomposite, a simple spectrophotometric method was employed to quantifythe drug. Absorbance at 274 nm has been correlated to amoxicillinconcentration in phosphate aqueous buffer using ε₂₇₄=1.2 mM⁻¹cm⁻¹ (Cary60 UV-V is spectrophotometer, Agilent Technologies). This extinctioncoefficient is consistent with other values found in the literature(ε_(274, ethanol)=1.4 mM⁻¹ cm⁻¹ and ε_(272, HCl 0.1M)=1.1 mM⁻¹cm⁻¹; TheMerck Index Online).

Amoxicillin Release.

An amount of loaded sol-gel polymer composite was first deposited at thevery bottom of a quartz cuvette, held at either T=37° C. or T=25° C. Thecuvette was then filled with a known volume of phosphate buffer (100 mMKH₂PO₄, 100 mM NaCl, pH=6.5). At this point, amoxicillin starteddiffusing out of the sol-gel. This release was monitored by followingthe absorbance of the solution above the sol-gel aliquot at 274 nm.Amoxicillin release (FIG. 8) was calculated by

Release %=(A/εl)·(n _(amox) /V)

where A is absorbance at 274 nm, ε is the extinction coefficient ofamoxicillin at 274 nm (1.2 mM⁻¹cm⁻¹), l is the path length of thecuvette, n_(amox) is the quantity of amoxicillin initially contained inthe sol-gel deposited to the cuvette bottom, and V is the total volumein the cuvette (i.e., V_(buffer)+V_(gel)). The curves on FIG. 8represent the average release from experiments performed on 3 sol-gelpolymer composite batches. The 37° C. curve (that is, physiologicaltemperature) depicted an early release behavior by the sol-gel, with 50%of the drug released over about 1 hour, followed by a sustained releaseuntil 100% of amoxicillin was released after approximately 6 hours,which would be beneficial to achieving quick and sustained blood levelsof the antimicrobial agent. At 25° C., the release was much slower, asonly about 20% of amoxicillin was released of the sol-gel polymercomposite after 5 hours. These results demonstrate that the loadedsol-gel formulation released its content following a fairly gradualrelease curve at physiological temperature. Furthermore, the releaserate was found to be positively correlated to the sample temperature.

TABLE 7 Comparison of the rheological properties of the loaded and theunloaded sol-gels. G′ G″ T (° C.) (Pa) (Pa) tan δ Unloaded 25 70 ± 40 30± 10 0.40 ± 0.10 (n = 6) 37 700 ± 200 50 ± 10 0.07 ± 0.02 Loaded 25 80 ±50 30 ± 10 0.37 ± 0.03 (n = 3) 37 500 ± 100 40 ± 10 0.09 ± 0.04

Antimicrobial Activity:

The antimicrobial activity of the amoxicillin loaded sol-gel polymercomposite was evaluated by the Kirby-Bauer Disc Susceptibility Test. Itsinhibition zone was evaluated and compared with that of the non-loadedgel.

An aliquot of 1 g of amoxicillin loaded F2 sol-gel, prepared asdescribed above, was spread on a 25 mm cellulose disc (Millipore). Thedisc was then deposited on a TSA II blood agar plate (Oxoid) that wasinoculated with 100 μL of Escherichia coli ATCC 25922 in broth cultureand diluted to match a 0.5 McFarland turbidity standard. The sameprocedure was used for the control experiment, which was carried outusing a non-loaded F2 sol-gel.

After 24 hours of incubation, an inhibition zone of d=42 mm (Table 8)was visible in the surroundings of the cellulose disc for the loadedgel, while the control exhibited no inhibition zone. With an inhibitionzone of 16 mm above disc size, the efficient antimicrobial action ofamoxicillin on E. coli ATCC 25922 is observed.

TABLE 8 Summary Kirby-Bauer Disk Susceptibility Test results. TInhibition zone (° C.) (mm) Unloaded 37 0 ± 0 (n = 1) Loaded 37 42 ± 2 (n = 3)

In summary, amoxicillin was shown by spectrophotometry to be entirelyreleased from the sol-gel polymer composite within 6 hours at 37° C.while solely 25% was released at 25° C. Finally, the sol-gel polymercomposite loaded with amoxicillin demonstrated a clear antimicrobialactivity compared to unloaded sol-gel as evaluated by Kirby-Bauer'ssusceptibility disc method.

Example 10 Evaluation of Sol-Gel Polymer Composite as Barrier toBacterial Migration

The purpose of the two tests in this study was to illustrate the abilityof the sol-gel polymer composite formulations to act as a barrieragainst bacterial migration in a simulated glass cow teat.

The sol-gel polymer composite formulations F1-F4 shown in Table 9 wereprepared as described hereinabove.

TABLE 9 Sol-gel polymer composite compositions. Sample Chitosan MethylSalt F1 1.8% 7.3% sodium pyrophosphate tetrabasic 0.05% F2   2%   4%potassium phosphate dibasic 0.7% F3   2%   4% sodium pyrophosphatetetrabasic 0.025%/ potassium phosphate dibasic 0.35% F4 1.8% 7.3%β-glycerophosphate disodium salt hydrate 3.6%The different sol-gel formulations can be defined by their respectiverheological properties in the form of Tan δ, G′ and G″ as presented inTable 10.

TABLE 10 Rheological properties, pH and appearance of sol-gel polymercomposite. Sample Tan δ 25/37° C. G′ (Pa) G″ (Pa) pH Appearance F11.59/0.04  4/672 7/23 6 Tan liquid F2 0.19/0.04 283/5798 52/239 6.59Tan, off- white F3 0.65/0.04  24/2742 16/75  6.29 Tan, off- white F4 0.1/0.04 398/5315 38/200 6.75 Yellowish, gelIn addition, a control motility test medium labeled “BAM 103” wasprepared by mixing tryptose 10.0 g/L and sodium chloride 5.0 g/L, thenhardened by the addition of agar 5.0 g/L.

Test 1—Evaluation of Impermeability of Sol-Gel Polymer Composite toBacteria:

Test 1 was based upon the standard bacteria motility test to test theability of bacteria to migrate through a gel medium comprising thesol-gel polymer composite samples. For this purpose, the motile bacteriaEscherichia coli ATCC 25922 was inoculated as a 1/10 dilution ofovernight culture in Tryptic Soy Broth (TSB) to match a 0.5 McFarlandturbidity standard. In a 15 mL polypropylene test tube, a 10 mL gel plugbeing investigated was first added, then a layer of Triphenyltetrazoliumchloride (TTC 0.5 g/L) was added on top of each test formulation. TTC isa bacteria-sensitive dye, which forms a red precipitate upon reductionin contact with bacteria.

The bacterial solution cultured overnight in TSB was added as a thirdlayer. 1 mL of this inoculum was added to the TTC and the test tube wasincubated for 24 and 48 hours at 37° C.

The ability of the bacteria to migrate in the sol-gel polymer compositeformulation is evaluated by the depth of red color measured from top tobottom of the lower sol-gel layer in the test tube. The test tubes wereexamined for color, which may spread from top to bottom depending onpotential bacterial migration. There was an initial red color thatappeared at the bacterial-gel interface due to partial penetration inthe medium (reduction of TTC). As presented in Table 11, the F1-F4sol-gel polymer composite formulations of the present disclosure do notallow bacterial penetration while BAM103 control allowed bacterialpenetration within 48 h. Bacterial penetration of the stiff agar controlwas observed only at the interface between the F1-F4 gels and tubes,where the thin interfacial water layer of the agar formed.

TABLE 11 Results of measured bacterial penetration (n = 3) intodifferent sol-gel polymer composite formulations and controls. Depth ofbacterial penetration (mm) time Sample 24 h incubation 48 h incubationF1 0 0 F2 0 0 F3 0 0 F4 0 0 BAM 103 48 ± 2 100  BAM 103 no 0 0inoculation Stiff Agar  0*  0* *no penetration inside the gel wasdetected but penetration at Agar-test tube interface was found to be upto 30 mm

The preceding experiment demonstrated the ability of the sol-gel polymercomposite formulations to be impermeable to motile bacteria.

Test 2—Evaluation of Sol-Gel Polymer Composite Barrier Properties in anArtificial Glass Cow Teat:

Test 2 was used to evaluate the ability of the sol-gel polymer compositeto prevent bacterial migration between two containers. For thisexperiment, a bottom, first container 1 was filled with nutrient brothand bacteria. A top second container 2 was filled with sterile nutrientbroth. The two containers were linked by a simulated glass teat (ø 26mm, 6 cm length, hole ø 2.6 mm) filled with the sol-gel polymercomposite formulation F3, prepared as described hereinabove. Theexperiment consisted of tracking the presence or absence of bacterialcontamination that may migrate upward into container 2 over time at 37°C.

The bacterial strains presented in Table 12 were selected for the testas a mixed culture that would be representative of a source of bovinemastitis.

TABLE 12 Strain selected for mixed culture in TSB as representativesource of bovine mastitis. Microorganism Response Motility Escherichiacoli ATCC 25922 Growth Positive Staphylococcus aureus ATCC 25923 GrowthNegative Klebsiella pneumoniae U 3023 Growth Negative

In this experiment, the adhesion between glass and the sol-gel polymercomposite plug appeared to be critical for barrier performance. Presenceof bubbles, uneven adhesion or successive sol-gel polymer compositetransition reduced performance by reducing glass-gel adhesion. Thisphenomenon was particularly significant at the start of the experiment,provoking a direct merge of the two containers. Tracking glass-geladhesion failure was performed by addition of Triphenyltetrazoliumchloride (TTC 0.5 g/L), a bacteria-sensitive dye, which forms a redprecipitate upon reduction in contact with bacteria.

The study of the ability of TTC—labeled F3 to maintain a bacterialbarrier property over time was evaluated. On Days 0, 2, 5, 6, 9, 12 and18, bacterial contamination was evaluated in both sterile andcontaminated media compartments. Observations were recorded on Days 0,3, 6, 14, and 15. Culture medium replacement and fresh inoculation wasadded to the contaminated compartment on Days 3, 6, 9, and 13. At Day12, signs of partial adhesion failure began to show a reduction ofglass-gel adhesion. On Days 12 and 17, adhesion failure at the glass cowteat interface with the F1-F4 sol-gel polymer composite sample wasobserved. On Day 15, there was a visual detection of contamination inthe upper container 2 above the gel plug (i.e., TTC dye diffusion showedred color in the initially non-contaminated compartment when thewell-adhered plug at Day 0 lost adhesion to the glass). At Day 17, theplug stopped to adhere completely to the glass. By Day 18, bacterialcontamination quantification by plate count proved sterility breach asthe loss of adherence to the glass allowed bacteria to migrate throughthe gel-glass interface leading to the contamination of container 2.

Knowing the high risk of adhesion breach of sol-gel to glass, this testis thus highly unfavorable to sol-gel performance. Until adhesionfailure, the actual performance reported for several tests between 1 and15 days of experiments and combined in Table 13 shows that F3 preventedbacterial contamination. Petroleum jelly/Paraffin Wax 1:5 control isbased on a hydrophobic plug that is known as fully impermeable tobacteria but the control also experienced adhesion issues with glasssimilar to those observed for the sol-gel composite. Results show thesimilar performance for this impermeable control and the sol-gel polymerformulations of the disclosure. The performance obtained for F3, between8 and 15 days during the ability to keep adherence to glass and act as abarrier to bacteria is significantly higher than those observed for thepermeable control BAM103 which cannot prevent bacterial contaminationafter 24 h. Thus, Test 2 provides evidence that the sol-gel compositeformulations of the disclosure possessed the ability to be an effectivebarrier against bacterial migration.

TABLE 13 Summary of barrier properties of different sol-gel and controlbased on 15 days of incubation experiments. Days before contaminationSample detected up to 15 Comment F1 13.5 ± 5   n = 3. Clear signs ofglass-gel adhesion failure prior to contamination. F2 8.5 ± 1  n = 2.Clear signs of glass-gel adhesion failure prior to contamination. F3 13± 5.5 n = 3. Clear signs of glass-gel adhesion failure prior tocontamination. F4 9 n = 1 BAM 103 1 n = 3 Petroleum 13 ± 7   n = 3.Clear signs of glass-Gel adhesion Jelly/Paraffin failure prior tocontamination. Wax 1:5 Orbeseal 15+ n = 1

In summary, Test 1 demonstrated the ability of the sol-gel polymercomposite formulations to prevent bacterial migration within 48 hours ascompared to a control gel BAM103 that allowed migration of motilebacteria. The results of the different sol-gel polymer compositeformulations showed a total impermeability to bacteria, thus preventingbacterial penetration into the gel. In Test 2, the barrier property inthe artificial glass cow teat against bacterial migration was shown tolast as long as adhesion between glass (simulated substrate) and thesol-gel plug was maintained. Performances were shown to be similar tothe impermeable control of paraffin/petroleum jelly and significantlylonger than the permeable control of BAM103. Since long-term retentionwas observed in the in vivo studies described in Examples 7 and 8, itcan be appreciated that the sol-gel polymer composite formulations ofthe disclosure will act as an effective teat sealant barrier againstboth motile and immotile bacterial migration during the dry period ofdairy cows until the sealants are physically removed.

Example 11 Evaluation of Impact of Inorganic Filler on Sol-Gel Rheology

Sol-gel polymer composite formulation F2 (1.5 L) was prepared asdescribed above in Example 1 and then mixed with filler (silicon dioxide(SiO₂) or nanocrystalline cellulose (NCC)) at various concentrations (1wt %, 5 wt %, and 20 wt %). The impact of the filler on density andrheological properties of the sol-gel polymer composite formulation wasevaluated. Results are shown in Table 14.

TABLE 14 Comparison of the rheological properties of sol-gel polymercomposite formulation with and without filler. Filler T (° C.) G′ (Pa)G″ (Pa) tan δ Density (g/ml) None 25 387.7 74.8 0.193 0.96 37 974.5 68.40.071 SiO₂ 25 194.3 66.0 0.340 1.03 1% 37 1067.4 65.9 0.062 SiO₂ 25235.4 70.2 0.298 1.11 5% 37 1134.4 72.8 0.065 SiO₂ 25 557.7 140.9 0.2531.09 20% 37 2089.0 153.6 0.074 NCC 25 196.4 66.2 0.337 1.08 1% 37 1067.866.6 0.063 NCC 25 312.0 99.8 0.320 1.08 5% 37 1712.5 122.7 0.072 NCC 25— — — 1.18 20% 37 — — —

The results show that the storage modulus (G′) increased by increasingthe amount of filler in the sol-gel polymer composite formulation,indicating that the higher the solid content in the formulation is, thestiffer the gel would be at 37° C. At 1 and 5 wt. % of SiO₂, the lossmodulus remained slightly the same compared to the original formulation(“None”). The same observation could be made for the NCC except for the5 wt % where the G″ increased at both temperatures compared to the mainmaterial. Concerning the tan δ at 25° C., it increased at 1 wt. % ofSiO₂ which indicates that gel is flowing better at room temperaturecompared to the original batch (“None”). It then decreased with theincrease of the filler content for the same reasons described previouslyregarding the solid content. The same behavior was seen for the NCC. Nogel transition was clearly observed with 20 wt. % NCC. Finally, theoriginal batch had lower density (0.96 g/ml) than water due to thepresence of air bubbles within the sol-gel. The addition of filler up to20 wt. % to the sol-gel formulation allowed the product to attain adensity of about 1.10 g/ml within the error margin of F2 formulationfree of air bubbles. In sum, the results show that addition of silicondioxide inorganic filler allows significant increase of G′ (up to aboutdouble) for both the sol and gel states without affecting the transitionfrom sol to gel and shear thinning of the formulation.

Example 12 Evaluation of Effect of Chitosan Degree of Deacetylation(DDA) on Sol-Gel Properties

Sol-gel polymer composite formulation F2 was prepared as described abovein Example 1 using chitosan having varying degrees of deacetylation (%DDA). Results are shown in Table 15.

When the chitosan % DDA in formulation F2 dropped below 76%, theformulation began to lose its sol-gel characteristics. In particular,the tan δ values observed at 25° C. and 37° C., and especially the tan δratio (tan δ at 25° C./tan δ at 37° C.), provides an indication andscale of the sol-gel transition. Upon transition to a low % DDA-sourcedchitosan (from 90% to 75.5% DDA), a noticeable decrease in F2performance was observed wherein the tan δ ratio decreased from 3.3-6.4to 1.2, as shown in Table 15 below.

TABLE 15 Comparative results of % DDA variation between lab-scale and 2L scale process. tanδ at tanδ at tanδ Scale % DDA 25° C. 37° C. ratio  50 g 90.0* 0.275 0.043 6.4 1.25 L 90.0* 0.227 0.047 4.8 1.25 L 90.0*0.253 0.076 3.3   50 g 75.5 0.140 0.113 1.2 1.25 L 75.5 0.177 0.144 1.2*% DDA was determined using a different batch of the same specificationof reagent-grade chitosan.

In order to explain the decrease in the sol-gel performance, atwo-factor Design of Experiment (DOE) was devised. Two parameters ofchitosan were considered, namely degree of deacetylation (% DDA) andmolecular weight (MW).

The most significant output factors and their expected values for anoptimal F2 formulation, while maintaining acceptable tan δ values at 37°C., are the following: Complex viscosity at 25° C.; tan δ at 25° C.; andoscillation stress of gelation.

Complex viscosity shall be low and tan δ at 25° C. values shall be highin order for the F2 formulation to exhibit the most “liquid” characteras possible; this would be expected to maximize scale-up processabilityfor the formulation. Oscillation stress of gelation shall be low inorder to increase the product's “syringeability”, ultimately forrepeated product delivery via syringe by the end user. DOE results aresummarized in Table 16.

TABLE 16 Summary of results from two-factor design of experiment (DOE).Onset of Chitosan Complex Oscillation Gelation MW Chitosan tanδ at tanδat viscosity stress Temp. (kDa) % DDA 25° C. 37° C. tanδ ratio (Pa · s)(Pa) (° C.) 84527 76.77% 0.27 0.07 3.9 256 218 28.8 55381  94.3% 0.370.07 5.3 140 150 31.0 467690 77.36% 0.27 0.08 3.4 375 217 34.0 40458097.60% 0.60 0.07 8.6 107 119 30.1

The results shown in Table 16 indicate that % DDA was the mostsignificant factor in decreasing complex viscosity, increasing tan δ at25° C., and decreasing formulation syringeability. The results of thisDOE also suggested that the high MW/high % DDA (404580 kDa, 97.6% DDA)chitosan produced an optimal formulation in terms of these properties.The resulting formulation yielded the least viscous (complex viscosity),most viscous liquid—as opposed to gel-like—nature (tan δ at 25° C.) andthe most syringeable formulation (oscillation stress) of all samplesexamined whilst maintaining a suitably viscous gel at 37° C. (tan δ at37° C.). The results suggest that, in some embodiments, chitosan havinga % DDA of about 77% or higher is required to obtain functional sol-gel.

In the foregoing, there has been provided a detailed description ofparticular embodiments of the present disclosure for the purpose ofillustration and not limitation. It is to be understood that all othermodifications, ramifications and equivalents obvious to those havingskill in the art based on this disclosure are intended to be includedwithin the scope of the disclosure as claimed.

What is claimed is:
 1. A product for preventing or treating a mammarydisorder in a female non-human animal, the product comprising aneffective amount of a sol-gel polymer composite.
 2. The productaccording to claim 1, wherein the sol-gel polymer composite compriseschitosan, a hydrophilic polymer, a gelation agent, and a suitablemedium.
 3. The product according to claim 2, wherein the medium isaqueous acetic acid.
 4. The product according to claim 2, wherein thechitosan is acylated.
 5. The product according to claim 4, wherein theacylated chitosan is carboxymethyl chitosan.
 6. The product according toclaim 2, wherein the hydrophilic polymer is a water-solublepolysaccharide.
 7. The product according to claim 2, wherein thehydrophilic polymer is selected from the group consisting of methylcellulose, polyvinyl acetate, polyvinyl acetate-acylate, hydroxypropylcellulose, ethyl hydroxyethyl cellulose, hyaluronic acid, nonionictriblock copolymer, polyethylene glycol, and sodium alginate.
 8. Theproduct according to claim 7, wherein the hydrophilic polymer is anonionic triblock copolymer and the nonionic triblock copolymer is apoloxamer.
 9. The product according to claim 2, wherein the gelationagent is a thermogelling element.
 10. The product according to claim 2,wherein the gelation agent is a salt selected from the group consistingof β-Glycerophosphate disodium hydrate, β-Glycerophosphate disodiumpentahydrate, sodium pyrophosphate tetrabasic, potassium phosphatedibasic trihydrate and a mixture thereof.
 11. The product according toclaim 2, wherein the sol-gel polymer composite further comprises areinforcing agent.
 12. The product according to claim 11, wherein thereinforcing agent is selected from the group consisting ofnanocrystalline cellulose, an inorganic clay, an organic clay, carbonblack, fumed silica, graphene, graphite, a nanocrystalline starch, ananoclay, graphene, a carbon nanotube, an organic nanoclay, anorganoclay, montmorillonite, bentonite, kaolinite, hectorite,halloysite, and calcium phosphate.
 13. The product according to claim 2,wherein the sol-gel polymer composite further comprises one or moreantimicrobial agents.
 14. The product according to claim 13, wherein theantimicrobial agent is selected from the group consisting of amacrolide, a cephalosporin, a lincosaminide antibiotic, afluoroquinolone, a tetracycline, a penicillin, a spectinomycin, asulfonamide, a chloramphenicol, a fluorinated synthetic analog ofthiamphenicol and a mixture thereof.
 15. The product according to claim14, wherein the antimicrobial agent is a cephalosporin and thecephalosporin is ceftiofur hydrochloride.
 16. A method of preventing ortreating a mammary disorder in a female non-human animal, comprisingadministering an effective amount of a sol-gel polymer composite to ateat, a teat canal or a teat cistern of the animal.
 17. A system forforming a physical barrier in the teat canal or cistern of a non-humananimal for the prevention or treatment of a mammary disorder wherein thesystem comprises a delivery device containing a sol-gel polymercomposite.
 18. The system according to claim 17, wherein the deliverydevice is a syringe.
 19. The system according to claim 18, wherein thesyringe contains a unit dose or multiple doses.
 20. The system accordingto claim 17, wherein the mammary disorder is mastitis.
 21. The systemaccording to claim 17, wherein the animal is a dairy livestock animal.22. The system according to claim 21, wherein the livestock animal is aheifer, a cow, a goat, a sheep or a water buffalo.
 23. The systemaccording to claim 17, wherein the sol-gel polymer composite compriseschitosan, a hydrophilic polymer, a gelation agent, and a suitablemedium.
 24. Use of a sol-gel polymer composite in the manufacture of amedicament for the prevention or treatment of a mammary disorder in anon-human animal.
 25. The use according to claim 24, wherein the mammarydisorder is mastitis.
 26. The use according to claim 24, wherein themedicament is employed for teat dipping.
 27. The use according to claim24, wherein the medicament is employed for intramammary infusion. 28.The use according to claim 24, wherein the animal is a dairy livestockanimal.
 29. The use according to claim 28, wherein the livestock animalis a heifer, a cow, a goat, a sheep or a water buffalo.
 30. The useaccording to claim 24, wherein the sol-gel polymer composite compriseschitosan, a hydrophilic polymer, a gelation agent, and a suitablemedium.
 31. The use according to claim 30, wherein the sol-gel compositefurther comprises a reinforcing agent.
 32. The use according to claim30, wherein the sol-gel composite further comprises one or moreantimicrobial agents.
 33. A sol-gel polymer composite comprisingchitosan, a hydrophilic polymer, and a gelation agent, in an acidicwater-based medium, the sol-gel polymer composite forming a strong solidin response to one or more physiological stimulus.
 34. The sol-gelpolymer composite according to claim 33, wherein the one or morephysiological stimulus is temperature of about 37° C.
 35. The sol-gelpolymer composite according to claim 33, wherein the sol-gel polymercomposite is capable of forming a solid in response to one or morephysiological stimulus without the addition of any other agents.
 36. Thesol-gel polymer composite according to claim 35, wherein the sol-gelpolymer solidifies rapidly in response to the one or more physiologicalstimulus.
 37. The sol-gel polymer composite according to claim 33,wherein the chitosan is carboxymethyl chitosan.
 38. The sol-gel polymercomposite according to claim 33, wherein the hydrophilic polymer isselected from the group consisting of methyl cellulose, polyvinylacetate, PVA-acylate, hydroxypropyl cellulose, ethyl hydroxylethylcellulose, a poloxamer, polyethylene glycol and sodium alginate.
 39. Thesol-gel polymer composite according to claim 33, wherein the gelationagent is a salt.
 40. The sol-gel polymer composite of claim 39, whereinthe salt is β-Glycerophosphate disodium salt or sodium pyrophosphatetetrabasic salt.
 41. The sol-gel polymer composite according to claim33, wherein the acidic water-based medium comprises 0.1M aqueous aceticacid.